Novel cytidine deaminase

ABSTRACT

Genes encoding novel proteins named AID (Activation-Induced cytidine Deaminase), having structural relationship to APOBEC-1, one of RNA editing enzymes, and having a cytidine deaminase activity similar to APOBEC-1, has been found by preparing cDNA libraries for mouse B cell clone CH12F3-2, which undergoes class switch recombination from IgM to IgA at an extremely high rate together with activation of cells by stimulation with cytokines, with and without stimulating with cytokine, and performing subtraction cloning using the libraries.

[0001] This application is a continuation-in-part of PCT/JP00/01918, filed Mar. 28, 2000, and claims priority from Japanese Patent Application No. 11-87192, filed Mar. 29, 1999; Japanese Patent Application No. 11/178999, filed Jun. 24, 1999; and Japanese Patent Application No. 11/371382, filed Dec. 27, 1999.

FIELD OF INVENTION

[0002] The present invention relates to novel proteins having a cytidine deaminase activity; DNAs and fragments thereof (cDNAs, genomic DNAs, and primer DNAs) encoding the proteins; expression vectors comprising the DNAs; transformants transformed with the expression vectors; antibodies reactive to the proteins or fragments thereof; cells producing the antibodies; and methods for identifying substances that regulate production of the proteins, transcription of genes encoding the proteins into mRNAs, or enzyme activities of the proteins.

BACKGROUND

[0003] The germinal center of mammals comprises a highly specialized microenvironment required for the final process of maturation towards antigen specific memory cells and long-lived plasma cells (Embo J., 16:2996-3006, 199; Semin. Immunol., 4:11-17, 1992). In the microenvironment, it is known that two fundamental editing of the immunoglobulin genes take place (J. Exp. Med., 173:1165-1175, 1991; Embo. J., 12:4955-4967, 1993; Adv. Exp. Med. Biol., 186:145-151, 1985; Nature, 342:929-931, 1989; Cell, 67:1121-1129).

[0004] One is the somatic hypermutation (Curr. Opin. Immunol., 7:248-254, 1995; Annu. Rev. Immunol., 14:441-457, 1996; Science, 244:1152-1157, 1989), a phenomenon in which extensive point mutation of exon genes encoding variable regions of immunoglobulin occurs. Accumulation of the point mutation leads to selection of B cells expressing high affinity immunoglobulins on their cell surface, accompanied by the affinity maturation of antibodies (Embo. J., 4:345-350, 1985; Proc. Natl. Acad. Sci. USA, 85:8206-8210, 1988). As the result, immunoglobulin genes are edited as new functional genes.

[0005] Another is the class switch recombination (CSR). In the recombination, effector functions of antibodies, such as complement fixation, are selected by exchanging exons encoding constant region of immunoglobulin heavy chain (Curr. Top. Microbiol. Immunol., 217:151-169, 1996; Annu. Rev. Immunol., 8:717-735, 1990).

[0006] These two types of genetic editing are very important for effective humoral immunoreaction to eliminate harmful microbes. The molecular mechanisms of the genetic phenomena have not yet elucidated despite the extensive studies for several decades.

[0007] The present inventors isolated mouse B cell clone, CH12F3-2, as a research tool to elucidate the molecular mechanism of class switch recombination of immunoglobulin. In the B cell line, class switch recombination (CSR) from IgM to IgA begins several hours after stimulation with IL-4, TGF-β, and CD40L, and finally, over 80% of the cells become IgA positive (Immunity, 9:1-10, 1998; Curr. Biol., 8:227-230, 1998; Int. Immunol., 8:193-201, 1996).

[0008] Using the mouse B cell clone CH12F3-2, the present inventors had reported that the breakpoints of CSR distribute not only in switch region (S region), characteristic repeated sequences, but also in the neighboring sequences (Curr. Biol., 8:227-230, 1998). However, the breakpoints were rarely seen in I exon and C exon, locating at upstream and downstream of S region, respectively. Also, according to the accumulated scientific evidence, it has been shown that the transcription of I exon and C exon and the splicing of the transcripts are essential for CSR (Cell, 73:1155-1164, 1993; Science, 259:984-987, 1993; Proc. Natl. Acad. Sci, USA, 90:3705-3709, 1993; Cell, 81:833-836, 1995).

[0009] This namely suggests that the transcripts are involved in CSR either directly or indirectly. Accordingly, the present inventors propose a theory that class switch is initiated by the recognition of DNA-RNA complex structure and not by the recognition of nucleotide sequences of switch region. This idea is further fortified by the fact that even in the case that Sa region is substituted with Sα region or Sγ region by introducing a mini-chromosome to above-mentioned mouse B cell clone CH12F3-2, CSR in the mini-chromosome efficiently occurs by stimuli of cytokines (Immunity, 9:1-10, 1998).

[0010] In plants and Protozoa, RNA editing, another type of genetic editing, is widely used as a mean for producing functional genes from limited genome (Cell, 81:833-836, 1995; Cell, 81:837-840, 1995). mRNA editing of many molecules such as mRNA of apolipoprotein B (apoB), AMPA receptors, Wilmstumor-1, a-galactosidase and neurofibroinatosis type-1, and tRNA-Asp, have been reported (Trends Genet., 12:418-424, 1996; Curr. Opin. Genet. Dev., 6:221-231, 1996). Although the molecular mechanism of mammalian RNA editing has not yet been elucidated, one performed by APOBEC-1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1) becomes understood by degrees (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993).

[0011] In apoB RNA editing, the first base C (cytosine) of codon CAA, which encodes glutamine, is converted to U (uridine), which alters the codon to UAA. As the result, in-frame stop codon is made in the apoB mRNA (J. Cell., 81:187-195, 1995:, J. Cell., 50:831-840, 1987; Science, 238:363-266, 1987). apoB-48 and apoB-100 are transcripts of edited mRNA and unedited mRNA of apoB, respectively, and these proteins possess totally different physiological functions for each other (J. Biol. Chem., 271:2353-2356, 1996).

[0012] In the site-specific RNA-editing, auxiliary factors are required (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993). In the absence of auxiliary factors, APOBEC-1 shows only a cytidine deaminase activity, possessing non-specific low affinity to RNA (J. Biol. Chem., 268:20709-20712, 1993; J. Cell., 81:187-195, 1995; J. Biol. Chem., 270:14768-14775, 1995; J. Biol. Chem., 270:14762-14767, 1995). The expression and activity of the auxiliary factors are found not only in organs with apoB mRNA editing, but also in organs with undetectable level of APOBEC-1 expression, or organs without apoB mRNA editing (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712, 1993; Nucleic Acids Res., 22:1874-1879, 1994; Proc Natl. Acad. Sci, USA, 91:8522-8526, 1994; J. Biol. Chem., 269:21725-21734, 1994).

[0013] The unexpected expression of the auxiliary factors involved in apoB mRNA editing suggests that the auxiliary factors may be involved in more general cellular function or other yet unknown RNA editing. Since there are possibilities that CSR and hypbermutation, which are genetic editing relating to immunoglobulin, may be accomplished by RNA editing, it is very interesting to elucidate whether RNA editing takes place or not in the genetic editing of immunoglobulin gene mentioned above.

SUMMARY

[0014] The present invention provides AID (Activation-Induced cytidine Deaminase), a novel cytidine deaminase having structural relationship to APOBEC-1, one of RNA editing enzyme, and involved in RNA editing in germinal center B cells, where genetic editing of immunoglobulin gene occurs, and a DNA encoding the enzyme.

[0015] The present inventors intensively searched for novel genes involved in class switch recombination (CSR), one of major genetic editing of immunoglobulin gene. As a result, by preparing cDNA libraries for mouse B cell clone CH12F3-2, in which class switch recombination from IgM to IgA is shown to occur at an extremely high rate together with activation of cells by stimulation with cytokines, with and without stimulating with cytokine, and performing subtraction cloning using the libraries, the present inventors found genes encoding mouse- and human-derived novel proteins named AID (Activation-Induced cytidine Deaminase), having structural relationship to APOBEC-1, one of RNA editing enzymes, and having a cytidine deaminase activity similar to APOBEC-1.

[0016] The AID protein in the present invention possesses features described below, and considered to be a very important RNA-modifying deaminase involved in regulating B cell activation, CSR of immunoglobulin gene, somatic hypermutation, and affinity maturation, which all are genetic editing specific to germinal center function:

[0017] (1) ORF of cDNA encoding AID protein comprises 198 amino acids, with 24 kDa calculated molecular weight (mouse: SEQ ID NO:2, and human: SEQ ID NO:8). Mouse AID protein shows approximately 28 kDa molecular weight by SDS-PAGE.

[0018] (2) The amino acid sequence of AID protein is 34% and 26% identical to APOBEC-1 (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1) at amino acid sequence level, for mouse and human derived protein respectively.

[0019] (3) AID protein has cytidine/deoxycytidine deaminase motif, which is the active center of the deaminase activity conserved in amino acid sequences of proteins belonging to cytosine nucleoside/nucleotide deaminase family.

[0020] (4) Cytidine deaminase motif of AID protein is allied with RNA editing deaminase subgroup.

[0021] (5) AID protein has Leucine-rich region considered to be important in protein-protein interaction, similar to APOBEC-1. Four leucines in leucine-rich region of the AID protein are conserved in leucine-rich region of APOBEC-1 in rabbit, rat, mouse and human.

[0022] (6) In the primary structure of AID protein, all amino acid residues reported to be necessary for APOBEC-1 to bind RNA (Phe66□Phe87□His61□Glu63 and Cys93) are conserved.

[0023] (7) AID protein has pseudoactive site domain in its C terminal for forming homodimer, similar to APOBEC-1 and ECDDA, an E. coli derived cytidine deaminase. There are possibilities that AID protein forms homodimer, or associates with other auxiliary proteins.

[0024] (8) AID protein shows a concentration-dependent cytidine deaminase activity. The activity can be inhibited dose dependently by tetrahydrouridine (THU), a specific inhibitor of cytidine deaminase. Also, a zinc chelator, 1,10-o-phenanthro line, inhibits the cytidine deaminase activity of AID protein while 1,7-o-phenanthroline, the inactive isomer, shows a weak inhibition. Thus, AID protein can considered to be a zinc-dependent cytidine deaminase as APOBEC-1.

[0025] (9) Strong expression of AID mRNA expression is seen in lymph nodes (mesenteric and amygdaline). Also, weak expression in spleen is seen.

[0026] (10) mRNA expression of AID protein is seen in a variety of lymphoid tissues (Peyer's patch, mesenteric lymph node, axillary lymph node, spleen, and bone marrow). Especially, notable expression is seen in peripheral lymphoid organs, such as lymphatic nodes and Peyer's patch. Contrariwise, expression in primary lymphoid organs is lower than the peripheral lymphoid organs.

[0027] (11) Expression of AID mRNA is at detection limit level without cytokines (IL-4, CD40L, TGF-β) stimulation in mouse B cell clone CH12F3-2, in which the cytokines stimulate class switch from IgM to IgA in the cells, whereas the expression is induced 3 hours after stimulation, and maximal expression is seen after 12 hours, with cytokine stimulation.

[0028] (12) AMD mRNA expression in mouse B cell clone CH12F3-2 can be induced strongly when stimulated with three cytokines, IL-4, CD40L and TGF-β, simultaneously, rather than stimulated with any one of them. Also, it can be considered that de novo protein synthesis is necessary for augmentation of AID mRNA expression, as the AID mRNA expression induction by cytokines in mouse B cell clone CH12F3-2 can be inhibited by cycloheximide, an protein synthesis inhibitor.

[0029] (13) In the in vitro test, an augmentation of AID mRNA expression can be seen when normal mouse spleen B cells are stimulated with LPS alone, LPS+IL-4, or LPS+TGF-β.

[0030] (14) In the in vivo test, when normal mice are immunized with sheep red blood cells (SRBC), a significant augmentation of AID mRNA expression can be seen 5 days after immunization, in which SRBC are known to induce clonal expansion, germinal center formation, and class switch recombination and affinity maturation of immunoglobulin gene.

[0031] (15) The in vivo augmentation of AID mRNA expression by SRBC immunization is specifically seen in splenic CD19 positive B cells.

[0032] (16) AID mRNA expression in lymphoid organs is specifically seen in the germinal center, enriched with B cells activated by antigen stimulation.

[0033] (17) Human AID gene locates at locus 12p13, close to the locus 12p13.1, where APOBEC-1 gene locates.

[0034] According to the characteristics described above, the AID protein of the present invention can be considered to have a function of regulating various biological mechanisms required for generation of antigen-specific immunoglobulins (specific antibodies), which eliminate non-self antigen (foreign antigen, self-reacting cells, etc.) that triggers various diseases. The mechanism for generation of immunoglobulin having high specificity to antigens includes germinal center functions such as activation of B cells, class switch recombination of immunoglobulin gene, somatic hypermutation, and affinity maturation. The AID protein of the present invention can be considered to be one of the enzymes that play an important role in the genetic editing occurring in germinal center B cells (e.g. class switch recombination and somatic mutation).

[0035] The dysfunction of the AID protein of the present invention can be the cause for the humoral immunodeficiency since it induces failure of germinal center B cell function, such as antigen-specific B cell activation, class switch recombination, and somatic mutation. Reversely, the hyperfunction of AID protein may induce allergy disease or autoimmune disease since it can cause inappropriate B cell activation and needless class switch recombination and somatic mutation.

[0036] Therefore, regulation of the function of AID protein and the gene encoding it enables preventing and treating various immunodeficiencies, autoimmune diseases, and allergies, which result from, for example, B cell dysfunctions (e.g. IgA deficiency, IgA nephropathy, γ globulinemia, hyper IgM syndrome, etc.) or class switch deficiency of immunoglobulin. Thus, the AID protein and the gene encoding the AID protein can be targets for the development of drugs for therapy of diseases mentioned above.

[0037] Examples of diseases whose onset prevention, symptom remission, therapy and/or symptomatic treatment effect is expected by regulating the function of the AID protein of the present invention or the gene encoding it include, for example, primary immunodeficiency syndrome with congenital disorder of immune system, mainly various immunodeficiencies considered to develop by B cell deficiency, decrease, or dysfunction (e.g., sex-linked agammaglobulinemia, sex-linked agammaglobulinemia with growth hormone deficiency, immunoglobulin deficiency with high IgM level, selective IgM deficiency, selective IgE deficiency, immunoglobulin heavy chain gene deletion, κ chain deficiency, IgA deficiency, IgG subclass selective deficiency, CVID (common variable immunodeficiency), infantile transient dysgammaglobulinemia, Rosen syndrome, severe combined immunodeficiency (sex-linked, autosomal recessive), ADA (adenosine deaminase) deficiency, PNP (purine nucleoside phosphorylase) deficiency, MHC class II deficiency, reticular dysplasia, Wiskott-Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome, chromosomal aberration, familial Ig hypermetabolism, hyper IgE syndrome, Gitlin syndrome, Nezelof syndrome, Good syndrome, osteodystrophy, transcobalamin syndrome, secretory bead syndrome, etc.), various diseases with antibody production deficiency that are secondary immunodeficiency syndrome with disorder of immune system caused by an acquired etiology (for example, AIDS, etc.), and/or various allergic diseases (e.g., bronchial asthma, atopic dermatitis, conjunctivitis, allergic rhinitis, allergic enteritis, drug-induced allergy, food allergy, allergic urticaria, glomerulonephritis, etc.).

[0038] Namely, the AID protein of the present invention, a fragment thereof, a DNA encoding the AID protein, a fragment thereof, and an antibody against the AID protein are useful as reagents for developing drugs for prevention and therapy of such diseases.

[0039] Also, the DNA itself is useful as an antisense drug regulating the function of AID gene at a gene level and in a use in gene therapy. The protein or the fragments thereof (e.g. enzyme active site) itself is useful as a drug.

[0040] Furthermore, a DNA comprising a complementary nucleotide sequence to an arbitrary partial nucleotide sequence in the nucleotide sequence of genomic DNA encoding AID protein of the present invention (especially human AID protein) is useful as a primer DNA for polymerase chain reaction (PCR).

[0041] An arbitrary partial nucleotide sequence of genomic DNA encoding the AID protein (especially human AID protein) of the present invention can be amplified by PCR using the primer DNA pair. For example, in the case that mutation or deletion of the nucleotide sequence of genomic DNA (especially exon) encoding AID protein is presumed to cause a certain immunodeficiency or an allergy, mutations and deletions in the genomic DNA can be identified by amplifying an arbitrary partial nucleotide sequence of genomic DNA encoding the AID protein obtained from tissue or cells of immunodeficiency or allergy patients by PCR using a pair of the primer DNA, by analyzing the presence and the size of PCR products, and the nucleotide sequence of the PCR products, and by comparing the nucleotide sequence with the corresponding nucleotide sequence in the genomic DNA encoding the AID protein derived from the normal human. That is to say, this method is capable of not only, for example, elucidating relationships between immunodeficiency or allergy and AID protein, but also, in the case where AID protein is the cause of onset of a sort of disease (e.g. immunodeficiency and/or allergy), diagnosing the diseases by the methods mentioned above.

[0042] Furthermore, an antibody reactive to the AID protein of the present invention or a fragment thereof is extremely useful as an antibody drug by regulating functions of the AID protein.

[0043] Furthermore, the gene (DNA), protein, and antibody of the present invention are useful as reagents for searching substrates (e.g. RNA, etc.) interacting (binding) with the protein (enzyme) of the present invention, or other auxiliary proteins associated with the protein of the present invention, and for developing drugs targeting the substrates and auxiliary proteins.

[0044] Also, model animals can be generated by disrupting (inactivating) the AID gene base on the genetic information on the AID protein derived from mammals (e.g. mouse, etc.), which is one embodiment of the DNA of present invention. By analyzing the physical, biological, pathological, and genetic features of the model animal, it is possible to elucidate functions of the genes and the proteins of the present invention.

[0045] Furthermore, by introducing normal human AID gene or mutant human AID gene (e.g. mutant human AID genes derived from immunodeficiency patients), which is one embodiment of the present invention, into the model animal whose endogenous gene has been disrupted, model animals having only normal or mutant human AID genes of the present invention can be generated. By administering drugs (compounds, antibodies, etc.) targeting the introduced human AID genes to the model animals, therapeutic effects of the drugs can be evaluated.

[0046] Furthermore, a method for identifying a substance that regulates production of the AID protein of the present invention or transcription of a gene encoding the AID protein into mRNA, or a substrate that inhibits the enzyme activity of the AID protein (e.g. cytidine deaminase activity) are extremely useful as means to develop drugs for therapy and prevention of various diseases (especially, immunodeficiency and/or allergy) in which the above-mentioned AID protein or AID gene is considered to be involved.

[0047] Thus, the present invention, for the first time, provides blow-mentioned DNAs (cDNAs, genomic DNAs, and an arbitrary fragment thereof), proteins, expression vectors, transformants, antibody pharmaceutical compositions, cells, the use of the DNA fragments as primer DNAs, and methods for screening.

[0048] (1) A DNA or a fragment thereof encoding a protein comprising the amino acid sequence of SEQ ID NO:2 or 8.

[0049] (2) The DNA or the fragment of (1), wherein the protein has a cytidine deaminase activity.

[0050] (3) A DNA or a fragment thereof comprising the nucleotide sequence of SEQ ID NO:1 or 7.

[0051] (4) A DNA or a fragment thereof comprising a nucleotide sequence of (a) or (b) below:

[0052] (a) a nucleotide sequence comprising the nucleotide residues 93 to 689 of SEQ ID NO:1 or

[0053] (b) a nucleotide sequence comprising the nucleotide residues 80 to 676 of SEQ ID NO:7.

[0054] (5) A DNA or a fragment thereof of (a) or (b) below:

[0055] (a) a DNA or a fragment thereof that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:2 and having a cytidine deaminase activity or

[0056] (b) a DNA or a fragment thereof that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:8 and having a cytidine deaminase activity.

[0057] (6) A protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO:2 or 8.

[0058] (7) A protein or a fragment thereof comprising substantially the same amino acid sequence as that of SEQ ID NO:2 or 8 and having a cytidine deaminase activity.

[0059] (8) A proteins of (a) or (b) below.

[0060] (a) a mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:2, and that has a cytidine deaminase activity or

[0061] (b) a mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:8, and that has a cytidine deaminase activity.

[0062] (9) An expression vector comprising the DNA or the fragment of any one of (1) to (5).

[0063] (10) A transformant transformed with the expression vector of (9).

[0064] (11) An antibodies or a portion thereof reactive to the protein of any one of (6) to (8) or to a fragment of the protein.

[0065] (12) The antibodies or the portion of (11), wherein the antibody is a monoclonal antibody.

[0066] (13) A pharmaceutical composition comprising the antibody or the portion of (11) or (12), and a pharmaceutically acceptable carrier.

[0067] (14) A cell producing a monoclonal antibody reactive to the protein of any one of (6) to (8) or to a fragment of the protein.

[0068] (15) The cell of (14), wherein the cell is a hybridoma obtained by fusing, with a mammal-derived myeloma cell, a non-human mammal-derived B cell that produces a monoclonal antibody.

[0069] (16) The cell of (15), wherein the cell is a transgenic cell transformed by introducing, into a cell, either or both of a DNA encoding a heavy chain of the monoclonal antibody and a DNA encoding a light chain of the monoclonal antibody.

[0070] (17) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (c) below:

[0071] (a) SEQ ID NO:9,

[0072] (b) SEQ ID NO:10, or

[0073] (c) SEQ ID NO:35.

[0074] (18) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (e) below:

[0075] (a) SEQ ID NO:11,

[0076] (b) SEQ ID NO:12,

[0077] (c) SEQ ID NO:13,

[0078] (d) SEQ ID NO:14, or

[0079] (e) SEQ ID NO:15.

[0080] (19) A DNA comprising a complementary nucleotide sequence to an arbitrary partial nucleotide sequence of a nucleotide sequence of any one of (a) to (h) below:

[0081] (a) SEQ ID NO:9,

[0082] (b) SEQ ID NO:10,

[0083] (c) SEQ ID NO:11,

[0084] (d) SEQ ID NO:12,

[0085] (e) SEQ ID NO:13,

[0086] (f) SEQ ID NO:14,

[0087] (g) SEQ ID NO:15, or

[0088] (h) SEQ ID NO:25.

[0089] (20) The DNA of (19), wherein the DNA comprises a nucleotide sequence of any one of (a) to (q) below:

[0090] (a) SEQ ID NO:18,

[0091] (b) SEQ ID NO:19,

[0092] (c) SEQ ID NO:20,

[0093] (d) SEQ ID NO:21,

[0094] (e) SEQ ID NO:22,

[0095] (f) SEQ ID NO:23,

[0096] (g) SEQ ID NO:24,

[0097] (h) SEQ ID NO:25,

[0098] (i) SEQ ID NO:26,

[0099] (j) SEQ ID NO:27,

[0100] (k) SEQ ID NO:28,

[0101] (l) SEQ ID NO:29,

[0102] (m) SEQ ID NO:30,

[0103] (n) SEQ ID NO:31,

[0104] (o) SEQ ID NO:32,

[0105] (p) SEQ ID NO:33, or

[0106] (q) SEQ ID NO:34.

[0107] (21) Use of the DNA of (19) or (20) as a primer DNA in polymerase chain reaction.

[0108] (22) Use of a pair of DNA of any one of (a) to (n) below as primer DNAs in polymerase chain reaction:

[0109] (a) a DNA comprising the nucleotide sequence of SEQ ID NO:31 and a DNA comprising the nucleotide sequence of SEQ ID NO:32,

[0110] (b) a DNA comprising the nucleotide sequence of SEQ ID NO:20 and a DNA comprising the nucleotide sequence of SEQ ID NO:22,

[0111] (c) a DNA comprising the nucleotide sequence of SEQ ID NO:21 and a DNA comprising the nucleotide sequence of SEQ ID NO:30,

[0112] (d) a DNA comprising the nucleotide sequence of SEQ ID NO:24 and a DNA comprising the nucleotide sequence of SEQ ID NO:25,

[0113] (e) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,

[0114] (f) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,

[0115] (g) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,

[0116] (h) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,

[0117] (i) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,

[0118] (j) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,

[0119] (k) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,

[0120] (l) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,

[0121] (m) a DNA comprising the nucleotide sequence of SEQ ID NO:33 and a DNA comprising the nucleotide sequence of SEQ ID NO:29, or,

[0122] (n) a DNA comprising the nucleotide sequence of SEQ ID NO:18 and a DNA comprising the nucleotide sequence of SEQ ID NO:19.

[0123] (23) A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of:

[0124] (a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein and

[0125] (b) (i) comparing the level of the AID protein produced by the cells cultured in the presence of the substance with the level of the MD protein produced by the cells cultured in the absence of the substance or

[0126] (ii) comparing the level of the AID protein-encoding mRNA transcribed in the cells cultured in the presence of the substance with the level of the AID protein-encoding mRNA transcribed in the cells cultured in the absence or the substance.

[0127] (24) A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of:

[0128] (a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein and a protein other than the AID protein, wherein transcription of a gene encoding the other protein into mRNA is dependent in the cells on the degree of a signal of transcription of the gene encoding the AID protein into mRNA and

[0129] (b) comparing the level of the other protein produced by the cells cultured in the presence of the substance with the level of the other protein produced by the cells cultured in the absence of the substance.

[0130] (25) The method of (23) or (24), wherein the cells are transgenic cells transformed with a gene encoding the protein.

[0131] (26) The method of (24), wherein the cells are transgenic cells transformed with a gene encoding the protein and a gene encoding the other protein.

[0132] (27) The method of (26), wherein the protein is a reporter protein.

[0133] (28) The method of (27), wherein comparison of the level of the other protein is comparison of the level of a signal generated by the reporter protein.

[0134] (29) The method of (27) or (28), wherein the reporter protein is luciferase.

[0135] (30) A method for identifying a substance that inhibits an enzyme activity of an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8, the method comprising the step of (a) or (b) below:

[0136] (a) culturing, separately in the presence and the absence of the substance, mammal-derived B cells or tissues comprising the B cells, and comparing enzyme activities of the AID protein in the B cells separately cultured or

[0137] (b) (i) administering the substance separately to an All) gene knockout mouse whose endogenous AID gene is inactivated so that transcription of the endogenous AID gene into mRNA is inhibited, and to a normal mouse and

[0138] (ii) comparing enzyme activities of the AID proteins in the B cells isolated from the respective mice.

[0139] (31) The method of (30), wherein the enzyme activity is a cytidine deaminase activity.

[0140] Hereafter, the present invention is explained in detail, by clarifying the terms used in the present invention and general methods for producing the proteins, DNAs, antibodies, and cells of the present invention.

[0141] The “protein or a fragment thereof” means a protein and a fragment thereof derived from a mammal such as human, bovine, sheep, pig, goat, rabbit, rat, hamster, guinea pig, mouse, and so on, preferably a protein or a fragment thereof derived from human, rabbit, rat, or mouse, and particularly preferably, a protein of a fragment thereof derived from human or mouse.

[0142] As a particularly preferred embodiment, it means any protein or a fragment thereof below.

[0143] (1) A protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO:2 or 8.

[0144] (2) A protein or a fragment thereof comprising substantially the same amino acid sequence as that of SEQ ID NO:2 or 8 and having a cytidine deaminase activity.

[0145] (3) A mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:2, and that has a cytidine deaminase activity.

[0146] (4) A mammal-derived protein that comprises an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7, that is homologous to a protein comprising the amino acid sequence of SEQ ID NO:8, and that has a cytidine deaminase activity.

[0147] Here, “having substantially the same amino acid sequence” means that a protein has an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in the amino acid sequence shown in the references are substituted, deleted, and/or modified, and that a protein has an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, are added to the amino acid sequence shown in the references.

[0148] The protein of the present invention includes monomer molecule, homodimer in which one strand binds to another strand comprising an identical amino acid sequence, heterodimer in which one strand binds to another strand comprising a different amino acid sequence, and oligomers such as trimer or tetramer.

[0149] Also, the “fragment of a protein” means an arbitrary partial sequence (fragment) in the amino acid sequence that the above-mentioned AID protein of the present invention comprises. For example, it includes an enzyme active site required for the AID protein to exert an enzyme activity represented by a cytidine deaminase activity, and an interaction site required for the AID protein to bind or associate with substrates (e.g. mRNA, etc.) or various auxiliary proteins.

[0150] Alphabetical triplet or single letter codes used to represent amino acids in the present specification or figures mean amino acids as follows:

[0151] (Gly/G), glycine; (Ala/A), alanine; (Val/V), valine; (Leu/L), leucine; (Ile/I), isoleucine; (Ser/S), serine; (Thr/T), threonine; (Asp/D), aspartic acid; (Glu/E), glutamic acid; (Asn/N), asparagines; (Gln/Q) glutamine; (Lys/K), lysine; (Arg/R), arginine; (Cys/C), cysteine; (Met/M), methionine; (Phe/F), phenylalanine; (Tyr/Y), tyrosine; (Trp/W), tryptophan; (His/H), histidine; (Pro/P), proline.

[0152] The proteins and fragments of the present invention can be produced by properly using, in addition to genetic engineering technique mentioned below, methods well known in the art, such as chemical synthesis, cell culture method, and so on, or their modified methods.

[0153] Also, the AID protein of the present invention can be produced as a recombinant fusion protein with other protein (e.g. GST (Glutathione S-transferase), etc.). In this case, the fusion protein is advantageous in that it can be extremely easily purified by affinity chromatography employing adsorbent on which other protein molecule binding specifically to GST is immobilized. Moreover, since various antibodies reactive to GST are provided, the quantification of the fusion protein can be simply carried out by immunoassay (e.g. ELISA, etc.) using the antibodies against GST.

[0154] The DNA of the present invention is a DNA encoding protein of the present invention and a fragment thereof, and it includes any nucleotide sequence encoding the protein of the present invention and includes both genomic DNAs and cDNAs. Also, the DNA includes any DNA composed of any codons as long as the codons encode identical amino acids.

[0155] Also, the DNA of the present invention includes a DNA encoding mammal AID protein, and, as a preferred embodiment, a DNA encoding mouse AID protein or human AID protein can be exemplified.

[0156] Examples of specific embodiments are as follows:

[0157] (1) A DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:2 or 8.

[0158] (2) The DNA of (1), wherein the protein has a cytidine deaminase activity.

[0159] (3) A DNA comprising the nucleotide sequences of SEQ ID NO:1 or 7.

[0160] (4) A DNA comprising the nucleotide residues 93 to 689 of SEQ ID NO:1.

[0161] (5) A DNA comprising the nucleotide residues 80 to 676 of SEQ ID NO:7.

[0162] (6) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:1 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:2 and having a cytidine deaminase activity.

[0163] (7) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:7 and that encodes a mammal-derived protein being homologous to a protein that comprises the amino acid sequence of SEQ ID NO:8 and having a cytidine deaminase activity.

[0164] (8) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (c) below:

[0165] (a) SEQ ID NO:9,

[0166] (b) SEQ ID NO:10, or

[0167] (c) SEQ ID NO:35.

[0168] (9) A genomic DNA or a fragment thereof comprising a nucleotide sequence of any one of (a) to (e) below:

[0169] (a) SEQ ID NO:11,

[0170] (b) SEQ ID NO:12,

[0171] (c) SEQ ID NO:13,

[0172] (d) SEQ ID NO:14, or

[0173] (e) SEQ ID NO:15.

[0174] (10) A DNA comprising a complementary nucleotide sequence to an arbitrary partial sequence of a nucleotide sequence of any one of (a) to (h) below:

[0175] (a) SEQ ID NO:9,

[0176] (b) SEQ ID NO:10,

[0177] (c) SEQ ID NO:11,

[0178] (d) SEQ ID NO:12,

[0179] (e) SEQ ID NO:13,

[0180] (f) SEQ ID NO:14,

[0181] (g) SEQ ID NO:15, or

[0182] (h) SEQ ID NO:35.

[0183] (11) A DNA comprising a nucleotide sequence of any one of (a) to (q) below:

[0184] (a) SEQ ID NO:18,

[0185] (b) SEQ ID NO:19,

[0186] (c) SEQ ID NO:20,

[0187] (d) SEQ ID NO:21,

[0188] (e) SEQ ID NO:22,

[0189] (f) SEQ ID NO:23,

[0190] (g) SEQ ID NO:24,

[0191] (h) SEQ ID NO:25,

[0192] (i) SEQ ID NO:26,

[0193] (j) SEQ ID NO:27,

[0194] (k) SEQ ID NO:28,

[0195] (l) SEQ ID NO:29,

[0196] (m) SEQ ID NO:30,

[0197] (n) SEQ ID NO:31,

[0198] (o) SEQ ID NO:32,

[0199] (p) SEQ ID NO:33, or,

[0200] (q) SEQ ID NO:34.

[0201] Furthermore, a DNA encoding a mutant protein or a fragment thereof obtained by substituting, deleting, and/or modifying multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, or by inserting multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids in the amino acid sequence constituting the above-defined AID protein of the present invention or a fragment thereof is included in the DNA of the present invention.

[0202] The term “under stringent conditions” used herein means, for example, the following conditions. For example, in the case of carrying out hybridization using a probe with not less than 50 bases in 0.9% NaCl, target temperature of causing 50% dissociation (Tm) can be calculated from the formula below, and the hybridization temperature can be set as the formula below.

Tm=82.3° C.+0.41′(G+C)%−500/n−0.61×(formamide)% (n means the number of bases of the probe)

Temperature=Tm−25° C.

[0203] Also, in the case of using a probe with not less than 100 bases (G+C=40 to 50%), the changes of Tm as (1) and (2) below can be used as the indicator.

[0204] (1) Every 1% mismatch decreases Tm by approximately 1° C.

[0205] (2) Every 1% formamide decreases Tm by 0.6 to 0.7° C.

[0206] Thus, the temperature condition in the case of combination of complete complementary strands can be set as below.

[0207] (A) 65 to 75° C. (without formamide)

[0208] (B) 35 to 45° C. (with 50% formamide)

[0209] The temperature condition in the case of combination of incomplete complementary strands can be set as below.

[0210] (A) 45 to 55° C. (without formamide)

[0211] (B) 35 to 42° C. (with 30% formamide)

[0212] In the case of using probes with not more than 23 bases, temperature can be 37° C., or the formula below can also be used as an indicator.

Temperature=2° C.×(number of A+T)+4° C.×(number of C+G)−5° C.

[0213] The DNA of the present invention can be a DNA obtained by any method. For example, the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining these methods.

[0214] As used herein, an “isolated nucleic acid” is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in random, uncharacterized mixtures of different DNA molecules, transfected cells, or cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.

[0215] The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. For example, the substantially pure polypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0216] The invention also includes a polypeptide, or fragment thereof, that differs from the corresponding sequence shown as SEQ ID NO:2 or 8. The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In one embodiment, the polypeptide includes an amino acid sequence at least about 60% identical to a sequence shown as SEQ ID NO:2 or 8, or a fragment thereof. Preferably, the polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:2 or 8 and has at least one cytidine deaminase function or activity described herein. Preferred polypeptide fragments of the invention are at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of the sequence shown as SEQ ID NO:2 or 8 and have at least one cytidine deaminase activity described herein. Or alternatively, the fragment can be merely an immunogenic fragment.

[0217] As used herein, “% identity” of two amino acid sequences, or of two nucleic acid sequences, is determined using the algorithm of Karlin and Altschul (PNAS USA 87:2264-2268, 1990), modified as in Karlin and Altschul, PNAS USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignment for comparison purposes GappedBLAST is utilized as described in Altschul et al (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention.

[0218] Accordingly, in one aspect, the invention provides an isolated or purified nucleic acid molecule that encodes a polypeptide described herein or a fragment thereof. Preferably, the isolated nucleic acid molecule includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO:1 or 7. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide sequence shown in SEQ ID NO:1 or 7. In the case of an isolated nucleic acid molecule which is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO:1 or 7, the comparison is made with the full length of the reference sequence. Where the isolated nucleic acid molecule is shorter that the reference sequence, e.g., shorter than SEQ ID NO:1 or 7, the comparison is made to a segment of the reference sequence of the same length (excluding any loop, required by the homology calculation).

[0219] The DNA encoding the protein of the present invention can be prepared by the usual methods: cloning cDNA from mRNA encoding the protein of the present invention, isolating genomic DNA and splicing it, chemical synthesis, and so on.

[0220] (1) cDNA can be cloned from mRNA encoding the protein of the present invention by, for example, the method described below.

[0221] First, the mRNA encoding the protein of the present invention is prepared from the above-mentioned tissues or cells expressing and producing the protein of the present invention. mRNA can be prepared by isolating total RNA by a known method such as guanidine-thiocyanate method (Chirgwin et al., Biochemistry, 18:5294, 1979), hot phenol method, or AGPC method, and subjecting it to affinity chromatography using oligo-dT cellulose or poly-U Sepharose.

[0222] Then, with the mRNA obtained as a template, cDNA is synthesized, for example, by a well-known method using reverse transcriptase, such as the method of Okayama et al (Mol. Cell. Biol. 2:161 (1982); Mol. Cell. Biol. 3:280 (1983)) or the method of Hoffman et al. (Gene 25:263 (1983)), and converted into double-stranded cDNA. A cDNA library is prepared by transforming E. coli with plasmid vectors, phage vectors, or cosmid vectors having this cDNA or by transfecting E. coli after in vitro packaging.

[0223] The plasmid vectors used in this invention are not limited as long as they are replicated and maintained in hosts. Any phage vector that can be replicated in hosts can also be used. Examples of usually used cloning vectors are pUC19, λgt10, λgt11, and so on. When the vector is applied to immunological screening as mentioned below, a vector having a promoter that can express a gene encoding the desired protein in a host is preferably used.

[0224] cDNA can be inserted into a plasmid by, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can be inserted into a phage vector by, for example, the method of Hyunh et al. (DNA cloning, a practical approach, 1, p.49 (1985)). These methods can be simply performed by using a commercially available cloning kit (for example, a product from Takara Shuzo). The recombinant plasmid or phage vector thus obtained is introduced into an appropriate host cell such as a prokaryote (for example, E. coli: HB101, DH5a, MC1061/P3, etc).

[0225] Examples of a method for introducing a plasmid into a host are, calcium chloride method, calcium chloride/rubidium chloride method and electroporation method, described in Molecular Cloning, A Laboratory Manual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)). Phage vectors can be introduced into host cells by, for example, a method in which the phage DNAs are introduced into grown hosts after in vitro packaging. In vitro packaging can be easily performed with a commercially available in vitro packaging kit (for example, a product from Stratagene or Amersham).

[0226] The identification of cDNA encoding protein, its expression being augmented depending on the stimulation of cytokines like AID protein of the present invention, can be carried out by for example suppression subtract hybridization (SSH)(Proc. Natl. Acad. Sci. USA, 93:6025-6030, 1996; Anal. Biochem., 240:90-97, 1996) taking advantage of suppressive PCR effect (Nucleic Acids Res., 23:1087-1088, 1995), using two cDNA libraries, namely, cDNA library constructed from mRNA derived from stimulated cells (tester cDNA library) and that constructed from mRNA derived from unstimulated cells (driver cDNA library).

[0227] The preparation of cDNA libraries required for subtraction cloning can be performed by using commercially available kit, for example, PCR-Select Subtraction Kit (CLONTECH, cat: K1804-1). The experiment can be performed according to the document of procedure accompanying in the kit.

[0228] An example of practical experimental procedure is listed below, briefly.

[0229] PolyA⁺ RNA is prepared from cells with or without stimulation with appropriate stimulant as previously reported method (Nucleic Acids Res., 26:911-918, 1998). Next, cDNA is prepared using reverse transcriptase from each polyA⁺ RNA samples, as is the commonly used method. cDNA prepared from stimulated cells is used as tester cDNA and that prepared from unstimulated cells as driver cDNA.

[0230] According to the previous report mentioned above and experimental manuals accompanying with kit, driver cDNA is added to tester cDNA to perform subtraction. The efficiency of subtraction is monitored by adding small amount of exogenous DNA as a control. After subtraction, the exogenous DNA is concentrated.

[0231] The subtracted cDNA is cloned into appropriate plasmid expression vector to construct a plasmid library by commonly used method.

[0232] Similar to the previous report, many colonies are screened by differential hybridization method (Nucleic Acids Res., 26:911-918, 1998; RINSYO-MEN-EKI, 29:451-459, 1997). Here, as the hybridization probes, tester cDNA and driver cDNA mentioned above labeled with radioisotope can be used. Clones containing the objective DNA and that containing exogenous DNA can be distinguished by hybridizing the exogenous DNA with replicant filters.

[0233] Objective cDNA or its fragment can be obtained by selecting clones giving strong signal against radiolabeled tester cDNA probe rather than radiolabeled driver cDNA probe.

[0234] Also, cDNA encoding the protein of the present invention can be accomplished by other general cDNA screening method.

[0235] For instance, cDNA or its fragment encoding the protein of the present invention cloned by subtraction cloning method mentioned above, or chemically synthesized oligonucleotides corresponding to amino acid sequence of the protein of the present invention, are labeled with ³²P to make probes, then by well-known colony hybridization method (Crunstein et al., Proc. Natl. Acid. Sci. USA, 72:3961, 1975) or plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.2.108, 1989), commercial or originally prepared cDNA libraries can be screened. Furthermore, a method to amplify DNA including cDNA encoding the protein of the present invention by PCR, by constructing a pair of PCR primer based on cDNA or its fragment encoding the protein of the present invention isolated by the subtraction cloning mentioned above, can be listed.

[0236] When a cDNA library prepared using a cDNA expression vector is used, the desired clone can be screened by the antigen-antibody reaction using an antibody against the desired protein. A screening method using PCR method is preferably used when many clones are subjected to screening.

[0237] The nucleotide sequence of the DNA thus obtained can be determined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci. USA, 74:560 (1977)) or the dideoxynucleotide synthetic chain termination method using phage M13 (Sanger et al. Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977)). The nucleotide sequence can be easily determined using a commercial DNA sequencer.

[0238] The whole or a part of the gene encoding the protein of the present invention can be obtained by excising the clone obtained as mentioned above with restriction enzymes and so on.

[0239] (2) Also, the DNA encoding the protein of the present invention can be isolated from the genomic DNA derived from the cells expressing the protein of the present invention as mentioned above by the following methods.

[0240] Such cells are solubilized preferably by SDS or proteinase K, and the DNAs are deproteinized by repeating phenol extraction. RNAs are digested preferably with ribonuclease. The DNAs obtained are partially digested with appropriate restriction enzymes, and the DNA fragments obtained are amplified with appropriate phage or cosmid to generate a library. Then, clones having the desired sequence are detected, for example, by using radioactively labeled DNA probes, and the whole or a portion of the gene encoding the protein of the present invention is obtained from the clones by excision with restriction enzymes, etc.

[0241] For example, cDNA encoding a human-derived protein can be obtained by preparing a cosmid library into which human genomic DNAs (chromosomal DNAs) are introduced (“Laboratory Manual Human Genome Mapping,” M. Hori and Y. Nakamura, eds., Maruzen), screening the cosmid library to obtain positive clones containing DNA corresponding to the coding region of the desired protein, and screening the above cDNA library using the coding region DNA excised from the positive clones as a probe.

[0242] Also, the present invention relates to any fragment of DNA (cDNA, genomic DNA, etc.) encoding AID protein (especially human AID protein) of the present invention described above. DNA with complementary nucleotide sequence to any nucleotide sequence of cDNA or genomic DNA is useful as a primer DNA in polymerase chain reaction (PCR). By PCR using a pair of the primer DNA, any partial nucleotide sequence of genomic DNA encoding AID protein (especially human AID protein) of the present invention can be amplified.

[0243] For instance, in the case that mutation or deletion of genomic DNA (especially exon) encoding the AID protein is presumed to cause a certain immunodeficiency or allergy, the existence of such the mutation or deletion can be analyzed by PCR described below.

[0244] (1) Prepare a pair of primers comprising complementary nucleotide sequence to any partial nucleotide sequence of genomic DNA encoding AID protein of the present invention.

[0245] (2) Amplify the objective partial nucleotide sequence of the genomic DNA using the pair of primers, using genomic DNA encoding AID protein obtained from tissue or cells of immunodeficiency or allergy patients as templates.

[0246] (3) Analyze the existence of PCR products and the nucleotide sequence of the PCR products, and identify the mutation and deletion in the genomic DNA by comparing the nucleotide sequence and corresponding nucleotide sequence of genomic DNA encoding AID protein derived from normal human.

[0247] Thus, the method described above can not only elucidate, for example, the relation between immunodeficiency and/or allergy and AID protein, but also be used for the diagnosis of a certain kind of disease, in the case that AID protein is the cause of the disease.

[0248] Examples of the nucleotide sequence of the primer DNA are as follows:

[0249] (1) A DNA comprising a complementary nucleotide sequence to an arbitrary partial sequence of a nucleotide sequence of any one of (a) to (h) below:

[0250] (a) SEQ ID NO:9,

[0251] (b) SEQ ID NO:10,

[0252] (c) SEQ ID NO:11,

[0253] (d) SEQ ID NO:12,

[0254] (e) SEQ ID NO:13,

[0255] (f) SEQ ID NO:14,

[0256] (g) SEQ ID NO:15, or

[0257] (h) SEQ ID NO:35.

[0258] (2) A DNA comprising a nucleotide sequence of any one of (a) to (q) below:

[0259] (a) SEQ ID NO:18,

[0260] (b) SEQ ID NO:19,

[0261] (c) SEQ ID NO:20,

[0262] (d) SEQ ID NO:21,

[0263] (e) SEQ ID NO:22,

[0264] (f) SEQ ID NO:23,

[0265] (g) SEQ ID NO:24,

[0266] (h) SEQ ID NO:25,

[0267] (i) SEQ ID NO:26,

[0268] (j) SEQ ID NO:27,

[0269] (k) SEQ ID NO:28,

[0270] (l) SEQ ID NO:29,

[0271] (m) SEQ ID NO:30,

[0272] (n) SEQ ID NO:31,

[0273] (o) SEQ ID NO:32,

[0274] (p) SEQ ID NO:33, or,

[0275] (q) SEQ ID NO:34.

[0276] Also, the present invention relates to the use of the above-mentioned DNA fragment as a primer DNA in polymerase chain reaction.

[0277] Examples of the combination of primer DNAs for PCR in diagnosis accomplished by PCR gene amplification and by analyzing it are as follows:

[0278] (1) a DNA comprising the nucleotide sequence of SEQ ID NO:31 and a DNA comprising the nucleotide sequence of SEQ ID NO:32,

[0279] (2) a DNA comprising the nucleotide sequence of SEQ ID NO:20 and a DNA comprising the nucleotide sequence of SEQ ID NO:22,

[0280] (3) a DNA comprising the nucleotide sequence of SEQ ID NO:21 and a DNA comprising the nucleotide sequence of SEQ ID NO:30,

[0281] (4) a DNA comprising the nucleotide sequence of SEQ ID NO:24 and a DNA comprising the nucleotide sequence of SEQ ID NO:25,

[0282] (5) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,

[0283] (6) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,

[0284] (7) a DNA comprising the nucleotide sequence of SEQ ID NO:23 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,

[0285] (8) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:27,

[0286] (9) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,

[0287] (10) a DNA comprising the nucleotide sequence of SEQ ID NO:26 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,

[0288] (11) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:28,

[0289] (12) a DNA comprising the nucleotide sequence of SEQ ID NO:34 and a DNA comprising the nucleotide sequence of SEQ ID NO:29,

[0290] (13) a DNA comprising the nucleotide sequence of SEQ ID NO:33 and a DNA comprising the nucleotide sequence of SEQ ID NO:29, or,

[0291] (14) a DNA comprising the nucleotide sequence of SEQ ID NO:18 and a DNA comprising the nucleotide sequence of SEQ ID NO:19.

[0292] Moreover, the present invention also relates to a recombinant vector comprising the DNA encoding the protein of the present invention. As a recombinant vector of the present invention, any vector can be used as long as it is capable of retaining replication or self-multiplication in each host cell of prokaryotic and/or eukaryotic cells, including plasmid vectors and phage vectors.

[0293] The recombinant vector can easily be prepared by ligating the DNA encoding protein of the present invention with a vector for recombination available in the art (plasmid DNA and bacteriophage DNA) by the usual method.

[0294] Specific examples of the vectors for recombination used are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples of phages are a bacteriophage such as λ phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.

[0295] An expression vector is useful for expressing the DNA encoding the protein of the present invention and for producing the protein of the present invention. The expression vector is not limited as long as it expresses the gene encoding the protein of the present invention in various prokaryotic and/or eukaryotic host cells and produces this protein. Examples thereof are pMAL C2, pEF-BOS (Nucleic Acids Res. 18:5322 (1990) and so on), pME18S (Experimental Medicine: SUPPLEMENT, “Handbook of Genetic Engineering” (1992) and so on), etc.

[0296] Also, the protein of the present invention can be produced as a fusion protein with other protein. It can be prepared as a fusion protein, for example, with GST (Glutathione S-transferase) by subcloning a cDNA encoding the protein of the present invention, for example, into plasmid pGEX4T1 (Pharmacia), by transforming E. coli DH5α, and by culturing the transformant.

[0297] When bacteria, particularly E. coli are used as host cells, an expression vector generally comprises, at least, a promoter/operator region, an initiation codon, the DNA encoding the protein of the present invention, termination codon, terminator region, and replicon.

[0298] When yeast, animal cells, or insect cells are used as hosts, an expression vector is preferably comprising, at least, a promoter, an initiation codon, the DNA encoding the protein of the present invention, and a termination codon. It may also comprise the DNA encoding a signal peptide, enhancer sequence, 5′- and 3′-untranslated region of the gene encoding the protein of the present invention, splicing junctions, polyadenylation site, selectable marker region, and replicon. The expression vector may also contain, if required, a gene for gene amplification (marker) that is usually used.

[0299] A promoter/operator region to express the protein of the present invention in bacteria comprises a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia, it preferably comprises Trp promoter, lac promoter, recA promoter, λPL promoter, b 1pp promoter, tac promoter, or the like. Examples of a promoter to express the protein of the present invention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and so on. When the host is Bacillus, examples thereof are SL01 promoter, SP02 promoter, penP promoter, and so on. When the host is a eukaryotic cell such as a mammalian cell, examples thereof are SV40-derived promoter, retrovirus promoter, heat shock promoter, and so on, and preferably SV-40 and retrovirus-derived one. As a matter of course, the promoter is not limited to the above examples. In addition, using an enhancer is effective for expression.

[0300] A preferable initiation codon is, for example, a methionine codon (ATG).

[0301] A commonly used termination codon (for example, TAG, TAA, TGA) is exemplified as a termination codon.

[0302] Usually, used natural or synthetic terminators are used as a terminator region.

[0303] A replicon means a DNA capable of replicating the whole DNA sequence in host cells, and includes a natural plasmid, an artificially modified plasmid (DNA fragment prepared from a natural plasmid), a synthetic plasmid, and so on. Examples of preferable plasmids are pBR322 or its artificial derivatives (DNA fragment obtained by treating pBR322 with appropriate restriction enzymes) for E. coli, yeast 2μ plasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, and such for mammalian cells.

[0304] An enhancer sequence, polyadenylation site, and splicing junction that are usually used in the art, such as those derived from SV40 can also be used.

[0305] A selectable marker usually employed can be used according to the usual method. Examples thereof are resistance genes for antibiotics, such as tetracycline, ampicillin, or kanamycin.

[0306] Examples of genes for gene amplification are dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene, etc.

[0307] The expression vector of the present invention can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA encoding the protein of the present invention, termination codon, and terminator region, to an appropriate replicon. If desired, appropriate DNA fragments (for example, linkers, restriction sites, and so on), can be used by the usual method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

[0308] Transformants of the present invention can be prepared by introducing the expression vector mentioned above into host cells.

[0309] Host cells used in the present invention are not limited as long as they are compatible with an expression vector mentioned above and can be transformed. Examples thereof are various cells such as wild-type cells or artificially established recombinant cells usually used in technical field of the present invention (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, and such), animal cells, or insect cells).

[0310]E. coli or animal cells are preferably used. Specific examples are E. coli (DH5α, TB1, HB101, and such), mouse-derived cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, and such), rat-derived cells (PC12, PC12h), hamster-derived cells (BHK, CHO, and such), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, and such), and human-derived cells (Hela, diploid fibroblast-derived cells, myeloma cells, and HepG2, and such).

[0311] An expression vector can be introduced (transformed (transfected)) into host cells by known methods.

[0312] Transformation can be performed, for example, according to the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, 69:2110 (1972)), protoplast method (Mol, Gen. Genet., 168:111 (1979)), or competent method (J. Mol. Biol., 56:209 (1971)) when the hosts are bacteria (E. coli, Bacillus subtilis, and such), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, 75:1927 (1978)), or lithium method (J. Bacteriol., 153:163 (1983)) when the host is Saccharomyces cerevisiae, the method of Graham (Virology, 52:456 (1973)) when the hosts are animal cells, and the method of Summers et al. (Mol. Cell. Biol., 3:2156-2165 (1983)) when the hosts are insect cells.

[0313] The protein of the present invention can be produced by cultivating transformants (in the following, this term includes transfectants) comprising an expression vector prepared as mentioned above in nutrient media.

[0314] The nutrient media preferably comprise carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of host cells (transformants). Examples of the carbon source are glucose, dextran, soluble starch, and sucrose, and examples of the inorganic or organic nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meet extract, soy bean cake, and potato extract. If desired, they may comprise other nutrients (for example, an inorganic salt (for example, calcium chloride, sodium dihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, and so on).

[0315] Cultivation is performed by a method known in the art. Cultivation conditions such as temperature, pH of the media, and cultivation time are selected appropriately so that the protein of the present invention is produced in large quantities.

[0316] Specific media and cultivation conditions used depending on host cells are illustrated below, but are not limited thereto.

[0317] When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi, liquid media comprising the nutrient source mentioned above are appropriate. The media with pH 5 to 8 are preferably used.

[0318] When the host is E. coli, examples of preferable media are LB media, M9 media (Miller et al. Exp. Mol. Genet., Cold Spring Harbor Laboratory, p.431 (1972)), and so on. Using these media, cultivation can be performed usually at 14 to 43° C. for about 3 to 24 hours with aeration and stirring, if necessary.

[0319] When the host is Bacillus, cultivation can be performed usually at 30 to 40° C. for about 16 to 96 hours with aeration and stirring, if necessary.

[0320] When the host is yeast, an example of media is Burkholder minimal media (Bostian, Proc. Natl. Acad. Sci. USA, 77:4505 (1980)). The pH of the media is preferably 5 to 8. Cultivation can be performed usually at 20 to 35° C. for about 14 to 144 hours with aeration and stirring, if necessary.

[0321] When the host is an animal cell, examples of media are MEM media containing about 5 to 20% fetal bovine serum (Science, 122:501 (1952)), DMEM media (Virology, 8:396 (1959)), RPMI1640 media (J. Am. Med. Assoc., 199:519 (1967)), 199 media (Proc. Soc. Exp. Biol. Med., 73:1 (1950)), and so on. The pH of the media is preferably about 6 to 8. Cultivation can be performed usually at about 30 to 40° C. for about 15 to 72 hours with aeration and stirring, if necessary.

[0322] When the host is an insect cell, an example of media is Grace's media containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 82:8404 (1985)). The pH thereof is preferably about 5 to 8. Cultivation can be performed usually at about 20 to 40° C. for 15 to 100 hours with aeration and stirring, if necessary.

[0323] The protein of the present invention can be produced by cultivating transformants, especially mammalian cells, as mentioned above and allowing them to secrete the protein into the culture supernatant.

[0324] A culture filtrate (supernatant) is obtained by a method such as filtration or centrifugation of the obtained culture, and the protein of the present invention is purified and isolated from the culture filtrate by methods commonly used in order to purify and isolate a natural or synthetic protein.

[0325] Examples of the isolation and purification method are a method utilizing solubility, such as salting out and solvent precipitation method; a method utilizing the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a method utilizing charges, such as ion exchange chromatography and hydroxylapatite chromatography; a method utilizing specific affinity, such as affinity column chromatography; a method utilizing the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and a method utilizing the difference in isoelectric point, such as isoelectric focusing.

[0326] When the protein of the present invention exists in the periplasm or cytoplasm of cultured transformants (for example, E. coli), first, the cells are harvested by a usual method such as filtration or centrifugation and suspended in appropriate buffer. After the cell wall and/or cell membrane of the cells and such are disrupted by the method such as lysis with sonication, lysozyme, and freeze-thawing, the membrane fraction comprising the protein of the present invention is obtained by the method such as centrifugation or filtration. The membrane fraction is solubilized with a detergent such as Triton-X100 to obtain the crude extract. Finally, the protein is isolated and purified from the crude extract by the usual method as illustrated above.

[0327] By using a DNA (cDNA or genomic DNA) encoding a human-derived AID protein included in the protein of the present invention, transgenic non-human mammals secreting the human AID protein in their body can be prepared. Namely, by integrating the human-derived DNA into an endogenous locus of non-human mammals (e.g. mouse), the human AID protein of the present invention encoded by the DNA is expressed and secreted in their body. The transgenic non-human mammals are included in the present invention.

[0328] The transgenic non-human mammals can be prepared according to the method usually used for producing a transgenic animal (for example, see “Newest Manual of Animal Cell Experiment”, LIC press, Chapter 7, pp.361-408, (1990)).

[0329] Specifically, for example, a transgenic mouse can be produced as follows. Embryonic stem cells (ES cells) obtained from normal mouse blastocysts are transformed with an expression vector in which the gene encoding the human AID protein of the present invention and a marker gene (for example, neomycin resistance gene) have been inserted in an expressible manner. ES cells in which the gene encoding the human AID protein of the present invention has been integrated into the endogenous gene are screened by a usual method based on expression of the marker gene. Then, the ES cells screened are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, 77:7380-7384 (1980); U.S. Pat. No. 4,873,191).

[0330] The blastocyst is transplanted into the uterus of another normal mouse as the foster mother. Then, founder mice are born from the foster mother. By mating the founder mice with normal mice, heterozygous transgenic mice are obtained. By mating the heterozygous transgenic mice with each other, homozygous transgenic mice are obtained according to Mendel's laws.

[0331] Also, so-called “knockout mouse” can be generated based on the nucleotide sequence of DNA encoding mouse AID protein included in the present invention. The “knockout mouse” in the present invention means the mouse in which the endogenous gene encoding the mouse AID protein of the present invention is knocked-out (inactivated). For example, it can be generated by positive-negative selection method applying homologous recombination (U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; Proc. Natl. Acad. Sci. USA, 86:8932-8935, 1989; Nature, 342:435-438, 1989; etc.), and such knockout mice are one embodiment of the present invention.

[0332] The “antibody” in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, and preferably a monoclonal antibody.

[0333] Specifically, it includes an antibody reactive to the above-mentioned protein of the present invention and a fragment thereof.

[0334] The “antibody” of the present invention also includes a natural antibody that can be prepared by immunizing mammals such as mice, rats, hamsters, guinea pigs, or rabbits with the protein of the present invention (including natural, recombinant, and chemically synthesized protein and cell), a fragment thereof, or a transformant highly expressing the protein of interest by recombinant technology mentioned above; a chimeric antibody and a humanized antibody (CDR-grafted antibody) that can be produced by recombinant technology; and a human monoclonal antibody that can be produced by using human antibody-producing transgenic animals.

[0335] The monoclonal antibody includes those having any one of the iso-types of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.

[0336] The polyclonal antibody (antiserum) or monoclonal antibody of the present invention can be produced by known methods. Namely, mammals, preferably, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, or bovine, or more preferably, mice, rats, hamsters, guinea pigs, or rabbits are immunized, for example, with an antigen mentioned above with Freund's adjuvant, if necessary. The polyclonal antibody can be obtained from the serum obtained from the animal so immunized. The monoclonal antibodies are produced as follows. Hybridomas are produced by fusing the antibody-producing cells obtained from the animal so immunized and myeloma cells incapable of producing autoantibodies. Then the hybridomas are cloned, and clones producing the monoclonal antibodies showing the specific affinity to the antigen used for immunizing the mammal are screened.

[0337] Specifically, the monoclonal antibody can be produced as follows. Immunizations are done by injecting or implanting once or several times the above-mentioned protein of the present invention, a fragment thereof, the cells that express the protein, and so on as an immunogen, if necessary, with Freund's adjuvant, subcutaneously, intramuscularly, intravenously, through the footpad, or intraperitoneally into mice, rats, hamsters, guinea pigs, or rabbits, preferably mice, rats or hamsters (including transgenic animals generated so as to produce antibodies derived from another animal such as the transgenic mouse producing human antibody ). Usually, immunizations are performed once to four times every one to fourteen days after the first immunization. Antibody-producing cells are obtained from the mammal so immunized in about one to five days after the last immunization.

[0338] Hybridomas that secrete a monoclonal antibody can be prepared by the method of Kohler and Milstein (Nature, 256:495-497 (1975)) and by its modified method. Namely, hybridomas are prepared by fusing antibody-producing cells contained in a spleen, lymph node, bone marrow, or tonsil obtained from the non-human mammal immunized as mentioned above, preferably a spleen, with myeloma cells without autoantibody-producing ability, which are derived from, preferably, a mammal such as mice, rats, guinea pigs, hamsters, rabbits, or humans, or more preferably, mice, rats, or humans.

[0339] For example, mouse-derived myeloma P3/X63-AG8.653 (653; ATCC No. CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3.; or human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.

[0340] Hybridoma clones producing monoclonal antibodies can be screened by cultivating the hybridomas, for example, in microtiter plates and by measuring the reactivity of the culture supernatant in the well in which hybridoma growth is observed, to the immunogen used for the immunization mentioned above, for example, by an enzyme immunoassay such as RIA and ELISA.

[0341] The monoclonal antibodies can be produced from hybridomas by cultivating the hybridomas in vitro or in vivo such as in the ascites of mice, rats, guinea pigs, hamsters, or rabbits, preferably mice or rats, more preferably mice and isolating the antibodies from the resulting the culture supernatant or ascites fluid of a mammal.

[0342] In vitro cultivation can be performed depending on the property of cells to be cultured, on the object of a test study, and on various culture, by using known nutrient media or any nutrient media derived from known basal media for growing, maintaining, and storing the hybridomas to produce monoclonal antibodies in the culture supernatant.

[0343] Examples of basal media are low calcium concentration media such as Ham′F12 medium, MCDB153 medium, or low calcium concentration MEM medium, and high calcium concentration media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basal media can contain, for example, sera, hormones, cytokines, and/or various inorganic or organic substances depending on the objective.

[0344] Monoclonal antibodies can be isolated and purified from the culture supernatant or ascites mentioned above by saturated ammonium sulfate precipitation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), affinity chromatography using anti-immunoglobulin column or protein A column.

[0345] Furthermore, monoclonal antibodies can be obtained in a large quantity by cloning a gene encoding a monoclonal antibody from the hybridoma, generating transgenic bovines, goats, sheep, or pigs in which the gene encoding the antibody is integrated in its endogenous gene using transgenic animal generating technique, and recovering the monoclonal antibody derived from the antibody gene from milk of the transgenic animals (Nikkei Science, No.4, pp.78-84 (1997)).

[0346] The “chimeric antibody” of the present invention means a monoclonal antibody prepared by genetic engineering, and specifically, a chimeric monoclonal antibody, for example, mouse/human chimeric antibody, whose variable region is a mouse immunoglobulin-derived variable region and whose constant region is a human immunoglobulin-derived constant region.

[0347] The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG, IgM, IgA, IgD, IgE, etc. The constant region of the recombinant chimeric monoclonal antibody of the present invention can be that of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG.

[0348] The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.

[0349] For example, mouse/human chimeric monoclonal antibody can be prepared, by referring to Experimental Medicine: SUPPLEMENT, 1.6, No.10 (1988); and Examined Published Japanese Patent Application (JP-B) No. Hei 3-73280. Namely, it can be prepared by ligating CH gene (C gene encoding the constant region of H chain) obtained from the DNA encoding human immunoglobulin to the downstream of active VH genes (rearranged VDJ gene encoding the variable region of H chain) obtained from the DNA encoding mouse monoclonal antibody isolated from the hybridoma producing the mouse monoclonal antibody, and by ligating the C_(L) gene (C gene encoding the constant region of L chain) obtained from the DNA encoding human immunoglobulin to the downstream of active V_(L) genes (rearranged VJ gene encoding the variable region of L chain) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma, and operably inserting those into the same or different vectors in an expressible manner, followed by transformation of host cells with the expression vector, and cultivation of the transformants.

[0350] Specifically, DNAs are first extracted from mouse monoclonal antibody-producing hybridoma by the usual method, digested with appropriate restriction enzymes (for example, EcoRI and HindIII), electrophoresed (using, for example, 0.7% agarose gel), and analyzed by Southern blotting. After the electrophoresed gel is stained, for example, with ethidium bromide and photographed, the gel is given marker positions, washed twice with water, and soaked in 0.25 M HCl for 15 minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minutes with gentle stirring. The DNAs are transferred to a filter for 4 hours following the usual method. The filter is recovered and washed twice with 2×SSC. After the filter is sufficiently dried, it is baked at 75° C. for 3 hours, treated with 0.1×SSC/0.1% SDS at 65° C. for 30 minutes, and then soaked in 3×SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65° C. for 3 to 4 hours.

[0351] Next, ³²P-labeled probe DNA and hybridization solution are added to the bag and reacted at 65° C. about 12 hours. After hybridization, the filter is washed under an appropriate salt concentration, reaction temperature, and time (for example, 2×SSC/0.1% SDS, room temperature, 10 minutes). The filter is put into a plastic bag with a little 2×SSC, and subjected to autoradiography after the bag is sealed.

[0352] Rearranged VDJ gene and VJ gene encoding H chain and L chain of mouse monoclonal antibody respectively are identified by Southern blotting mentioned above. The region comprising the identified DNA fragment is fractionated by sucrose density gradient centrifugation and inserted into a phage vector (for example, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.). E. coli (for example, LE392, NM539, etc.) are transformed with the phage vector to generate a genomic library. The genomic library is screened by plaque hybridization such as the Benton-Davis method (Science, 196:180-182 (1977)) using appropriate probes (H chain J gene, L chain (κ) J gene, etc.) to obtain positive clones comprising rearranged VDJ gene or VJ gene respectively. By making the restriction map and determining the nucleotide sequence of the clones obtained, it is confirmed that genes comprising the desired, rearranged V_(H) (VDJ) gene or V_(L) (VJ) gene have been obtained.

[0353] Separately, human CH gene and human CL gene used for chimerization are isolated. For example, when a chimeric antibody with human IgG1 is produced, Cγ₁ gene is isolated as a C_(H) gene, and Cκ gene is also isolated as a C_(L) gene, are isolated. These genes can be isolated from human genomic library with mouse Cγ₁ gene and mouse Cκ gene, corresponding to human Cγ₁ gene and human Cκ gene, respectively, as probes, taking advantage of the high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human immunoglobulin gene.

[0354] Specifically, DNA fragments comprising human Cκ gene and an enhancer region are isolated from human λ Charon 4A HaeIII-AluI genomic library (Cell, 15:1157-1174 (1978)), for example, using a 3 kb HindIII-BamHI fragment from clone Ig146 (Proc. Natl. Acad. Sci. USA, 75:4709-4713 (1978)) and a 6.8 kb EcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA, 78:474-478 (1981)) as probes. In addition, for example, after human fetal hepatocyte DNA is digested with HindIII and fractioned by agarose gel electrophoresis, a 5.9 kb fragment is inserted into λ788 and then human Cγ₁ gene is isolated with the probes mentioned above.

[0355] Using mouse V_(H) gene, mouse V_(L) gene, human C_(H) gene, and human C_(L) gene so obtained, and taking promoter region and enhancer region into consideration, human C_(H) gene is inserted downstream of mouse V_(H) gene and human C_(L) gene is inserted downstream of mouse V_(L) gene in an expression vector such as pSV2gpt or pSV2neo with appropriate restriction enzymes and DNA ligase following the usual method. In this case, chimeric genes of mouse V_(H) gene/human C_(H) gene and mouse V_(L) gene/human C_(L) gene can be respectively inserted into a same or different expression vector.

[0356] Chimeric gene-inserted expression vector(s) thus prepared are introduced into myeloma cells (e.g., P3X63 Ag8 653 cells or SP210 cells) that do not produce antibodies by the protoplast fusion method, DEAE-dextran method, calcium phosphate method, or electroporation method. The transformants are screened by cultivating in a medium containing a drug corresponding to the drug resistance gene inserted into the expression vector and, then, cells producing desired chimeric monoclonal antibodies are obtained.

[0357] Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened.

[0358] The “humanized antibody (CDR-grafted antibody)” of the present invention is a monoclonal antibody prepared by genetic engineering and specifically means a humanized monoclonal antibody wherein a portion or the whole of the complementarity determining regions of the hyper-variable region are derived from those of the hyper-variable region from mouse monoclonal antibody, the framework regions of the variable region are derived from those of the variable region from human immunoglobulin, and the constant region is derived from that from human-immunoglobulin.

[0359] The complementarity determining regions of the hyper-variable region exists in the hyper-variable region in the variable region of an antibody and means three regions which directly binds, in a complementary manner, to an antigen (complementarity-determining residues, CDR1, CDR2, and CDR3). The framework regions of the variable region mean four comparatively conserved regions intervening upstream, downstream or between the three complementarity-determining regions (framework region, FR1, FR2, FR3, and FR4).

[0360] In other words, a humanized monoclonal antibody means that in which the whole region except a portion or the whole region of the complementarity determining regions of the hyper-variable region of a mouse monoclonal antibody has been replaced with their corresponding regions derived from human immunoglobulin.

[0361] The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG, IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonal antibody in the present invention can be that from human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. The framework regions of the constant region derived from human immunoglobulin are not particularly limited.

[0362] The humanized monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.

[0363] For example, a recombinant humanized monoclonal antibody derived from mouse monoclonal antibody can be prepared by genetic engineering, referring to Published Japanese Translations of PCT International Publication No. Hei 4-506458 and Unexamined Published Japanese Patent Application (JP-A) No. Sho 62-296890. Namely, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated from hybridomas producing mouse monoclonal antibody, and human H chain gene encoding the whole region except human H chain CDR corresponding to mouse H chain CDR mentioned above and human L chain gene encoding the whole region except human L chain CDR corresponding to mouse L chain CDR mentioned above are isolated from human immunoglobulin genes.

[0364] The mouse H chain CDR gene(s) and the human H chain gene(s) so isolated are inserted, in an expressible manner, into an appropriate vector so that they can be expressed. Similarly, the mouse L chain CDR gene(s) and the human L chain gene(s) are inserted, in an expressible manner, into another appropriate vector so that they can be expressed. Alternatively, the mouse H chain CDR gene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human L chain gene(s) can be inserted, in an expressible manner, into the same expression vector so that they can be expressed. Host cells are transformed with the expression vector thus prepared to obtain transformants producing humanized monoclonal antibody. By cultivating the transformants, desired humanized monoclonal antibody is obtained from culture supernatant.

[0365] The “human antibody” used in the present invention is immunoglobulin in which the entire regions comprising the variable and constant region of H chain, and the variable and constant region of L chain constituting immunoglobulin are derived from the genes encoding human immunoglobulin.

[0366] The human antibody can be produced in the same way as the production method of polyclonal or monoclonal antibodies mentioned above by immunizing, with an antigen, a transgenic animal which for example, at least human immunoglobulin gene(s) have been integrated into the locus of a non-human mammal such as a mouse by the usual method.

[0367] For example, a transgenic mouse producing human antibodies is prepared by the methods described in already published literatures (Nature Genetics, 7:13-21 (1994); Nature Genetics, 15:146-156 (1997); JP-WA Hei 4-504365; WO94/25585; Nikkei Science, No.6, pp.40-50 (1995); WO94/25585; Nature, 368:856-859 (1994); JP-WA No. Hei 6-500233).

[0368] The “portion of an antibody” used in the present invention means a partial region of the antibody, and preferably the monoclonal antibody of the present invention as mentioned above, and specifically, means F(ab′)₂, Fab′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domain antibody) (Exp. Opin. Ther. Patents, 6, No.5, pp.441-456 (1996)).

[0369] “F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and means an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which an L chain composed of V_(L) (L chain variable region) and C_(L) (L chain constant region), and an H chain fragment composed of V_(H) (H chain variable region) and C_(H)γ1 (γ1 region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond. Each of these two homologous antibody fragments is called Fab′. Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fab′ are connected at the hinge region. This antibody fragment is called F(ab′)₂.

[0370] The “cell producing a monoclonal antibody reactive to a protein or a fragment thereof” of the present invention means any cell producing the above-described monoclonal antibody of the present invention.

[0371] More specifically, the following is included:

[0372] (1) B cells that are obtained by immunizing the non-human mammals with the above-mentioned protein of the present invention, a fragment thereof, or the cells producing the protein and that produce a monoclonal antibody reactive to the protein of the present invention or a fragment thereof.

[0373] (2) The above-mentioned hybridomas (fused cell) prepared by fusing the thus-obtained B cells producing the antibody with myeloma cells derived from mammals.

[0374] (3) Monoclonal antibody-producing transformants obtained by transforming other cells than the monoclonal antibody-producing B cells and hybridomas with genes encoding the monoclonal antibody isolated from the monoclonal antibody-producing B cells or hybridomas (either the heavy chain-encoding gene or the light chain-encoding gene, or both).

[0375] The monoclonal antibody-producing transformants of (3) mean recombinant cells producing a recombinant monoclonal antibody produced by B cells of (1) or hybridomas of (2). These antibody producing-transformants can be produced by the method as used for producing the above-described chimeric monoclonal antibody and humanized monoclonal antibody.

[0376] The “pharmaceutical composition” used herein means a pharmaceutical composition comprising of any of the protein, fragment thereof, antibody, or portion thereof defined hereinabove, and a pharmaceutically acceptable carrier.

[0377] The “pharmaceutically acceptable carrier” includes an excipient, a diluent, an expander, a disintegrating agent, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a colorant, a sweetener, a viscosity-increasing agent, a flavor, a dissolving agent, or other additives. Using one or more of such carriers, a pharmaceutical composition can be formulated into tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions, or syrups. The pharmaceutical composition can be administered orally or parenterally. Other forms for parenteral administration include a solution for external application, suppository for rectal administration, and pessary, prescribed by the usual method, which comprises one or more active ingredient.

[0378] The dosage can vary depending on the age, sex, weight, and symptoms of a patient, effect of treatment, administration route, period of treatment, or the kind of active ingredient (protein or antibody mentioned above) contained in the pharmaceutical composition. Usually, the pharmaceutical composition can be administered to an adult in a dose of 10 μg to 1000 mg (or 10 μg to 500 mg) per one administration. Depending on various conditions, the lower dosage may be sufficient in some cases, and a higher dosage may be necessary in other cases.

[0379] In particular, the injection can be produced by dissolving or suspending the antibody in a non-toxic, pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injections by adjusting the concentration to 0.1 μg antibody/ml carrier to 10 mg antibody/ml carrier. The injection thus produced can be administered to a human patient in need of treatment in a dose of 1 μg to 100 mg/kg body weight, preferably 50 μg to 50 mg/kg body weight, once or more times a clay. Examples of administration routes are medically appropriate administration routes such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection, preferably intravenous injection.

[0380] The injection can also be prepared into a non-aqueous diluent (for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and alcohols such as ethanol), suspension, or emulsion.

[0381] The injection can be sterilized by filtration with a bacteria-non-penetrable filter, by mixing bacteriocide, or by irradiation. The injection can be prepared at the time of use. Namely, it is freeze-dried to make a sterile solid composition, and can be dissolved in sterile distilled water for injection or another solvent before use.

[0382] The pharmaceutical composition of the present invention is useful as a drug for preventing and treating, for example, primary immunodeficiency syndrome with congenital disorder of immune system, mainly immunodeficiency considered to develop by B lymphocyte deficiency, decrease, or dysfunction (e.g., sex-linked agammaglobulinemia, sex-linked agammaglobulinemia with growth hormone deficiency, immunoglobulin deficiency with high IgM level, selective IgM deficiency, selective IgE deficiency, immunoglobulin heavy chain gene deletion, κ chain deficiency, IgA deficiency, IgG subclass selective deficiency, CVID (common variable immunodeficiency), infantile transient dysgammaglobulinemia, Rosen syndrome, severe combined immunodeficiency (sex-linked, autosomal recessive), ADA (adenosine deaminase) deficiency, PNP (purine nucleoside phosphorylase) deficiency, MHC class II deficiency, reticular dysplasia, Wiskott-Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome, chromosomal aberration, familial Ig hypermetabolism, hyper IgE syndrome, Gitlin syndrome, Nezelof syndrome, Good syndrome, osteodystrophy, transcobalamin syndrome, secretory bead syndrome, etc.), various diseases with antibody production deficiency that are secondary immunodeficiency syndrome with disorder of immune system caused by an acquired etiology (for example, AIDS, etc.), and/or various allergic diseases (e.g., bronchial asthma, atopic dermatitis, conjunctivitis, allergic rhinitis, allergic enteritis, drug-induced allergy, food allergy, allergic urticaria, glomerulonephritis, etc.), and for reliving condition due to various immunodeficiency with the diseases.

[0383] The DNA of the present invention described above, namely, “DNA comprising any partial nucleotide sequence of SEQ ID NO:7, from SEQ ID NO:9 to SEQ ID NO:15, SEQ ID NO:35, those with partial chemical modification, DNA comprising complementary nucleotide sequences to the partial sequence, or those with partial chemical modification” are included.

[0384] Here, the “partial nucleotide sequence” means the partial nucleotide sequence comprising any number of bases at any region included in any nucleotide sequence listed in SEQ ID NO:7, from SEQ ID NO:9 to SEQ ID NO:15, or SEQ ID NO:35.

[0385] The DNA is useful as probes in DNA hybridization or RNA hybridization procedures. In the purpose of using the DNA as a probe, continuous nucleotide sequence of over 20 bases, preferably continuous nucleotide sequence of over 50 bases, more preferably over 100 bases, much more preferably over 200 bases, especially preferably over 300 bases, can be listed as the partial nucleotide sequences.

[0386] Also, the DNA described above as mentioned before, are useful as primers for PCR. In the purpose of using the DNA as PCR primers, continuous partial nucleotide sequence of from 5 to 100 bases, preferably from 5 to 70 bases, more preferably from 5 to 50 bases, much more preferably from 5 to 30 bases, can be listed as the partial nucleotide sequences.

[0387] Moreover, the DNA described above are useful as antisense drug. The DNA, hybridizing to a DNA or an RNA encoding the AID protein of the present invention, can inhibit transcription of the DNA to mRNA or translation of the mRNA into the protein.

[0388] In purpose of using above-mentioned DNA to antisense drug, the partial nucleotide sequence consists of 5 to 100 consecutive nucleotides, preferably 5 to 70 consecutive nucleotides, more preferably 5 to 50 consecutive nucleotides, and still more preferably 5 to 30 consecutive nucleotides.

[0389] When the DNA is used as an antisense DNA pharmaceutical, the DNA sequence can be modified chemically in part for extending the half-life (stability) of the blood concentration of the DNA administered to patients, for increasing the intracellular-membrane permeability of the DNA, or for increasing the degradation resistance or the absorption of the orally administered DNA in the digestive organs. The chemical modification includes, for example, the modification of the phosphate bonds, the riboses, the nucleotide bases, the sugar moiety, the 3′ end and/or the 5′ end in the structure of the oligonucleotide DNA.

[0390] The modification of phosphate bond includes, for example, the conversion of one or more of the bonds to phosphodiester bonds (D-oligo), phosphorothioate bonds, phosphorodithioate bonds (S-oligo), methyl phosphonate (MP-oligo), phosphoroamidate bonds, non-phosphate bonds or methyl phosphonothioate bonds, or combinations thereof. The modification of the ribose includes, for example, the conversion to 2′-fluororibose or 2′-O-methylribose. The modification of the nucleotide base includes, for example, the conversion to 5-propynyluracil or 2-aminoadenine.

[0391] Also, another one of the present invention relates to “methods of identifying substances regulating the production of the AID protein of the present invention or the transcription of the gene encoding AID protein to mRNA”. The method of the present invention is namely “the method of screening of drugs capable of regulating functions of AID protein or AID gene”.

[0392] As the cells used in the method of the present invention, any cells, as long as capable of producing AID protein of the present invention, can be used. For instance, native cells (preferably of mouse or human), transgenic cells transformed with a gene encoding AID protein of the present invention, cells introduced with RNA encoding AID protein of the present invention, etc., can be listed.

[0393] As the host cells used for preparing the transgenic cells, various cells, mentioned in the part explaining in detail on the method of expressing the protein of the present invention using the DNA of the protein described above, can be used.

[0394] For instance, various cells such as naturally established cells or artificially established transgenic cells (e.g. bacteria (Escherichia, Bacillus), yeast (Saccharomyces, Pichia), animal cells and insect cells) can be exemplified.

[0395] Preferably, animal cells, namely, cells derived from mouse (COP, L, C127, Sp2/0, NS-1, or NIH3T3, etc.), cells derived from rat (PC12, PC12h, etc.), cells derived from hamster (BHK, and CHO, etc.), cells derived from monkey (COS1, COS3, COS7, CV1, and Velo, etc.), and cells derived from human (Hela, cells derived from diploid fibroblast, HEK293 cells, myeloma cells, and Namalwa, etc.) can be exemplified.

[0396] The “substance” in the present invention means natural substance existing in the nature and any substance prepared artificially. The substances can be grouped into “peptidic substance” and “non-peptidic substance”.

[0397] As the “non-peptidic substance”, “DNA comprising partial nucleotide sequence, or chemically modified DNA derived from it” that are useful as antisense drug as described above, “antisense RNA” with similar structural and pharmacological property to the antisense DNA, or any chemically synthesized “compounds” can be exemplified. The “compounds” herein means compounds excepting DNA, RNA, and peptidic substances. Namely, compounds with molecular weight of smaller than from 100 to approximately 1000, preferably compounds with molecular weight of from 100 to 800, more preferably molecular weight of from 100 to 600, can be exemplified.

[0398] As the “peptidic substance”, antibodies already described above in detail (preferably monoclonal antibodies, more preferably recombinant antibodies or human monoclonal antibodies), oligopeptides, or chemically modified substance derived from them can be exemplified. Examples of an oligopeptide are a peptide comprising 5 to 30 amino acids, preferably 5 to 20 amino acids. The chemical modification can be designed depending on various purposes, for example, the increased half-life in blood in the case of administering in vivo, or the increased tolerance against the degradation or increased absorption in digestive tract at the oral administration.

[0399] Methods described in from (24) to (28) above includes so-called reporter gene assay, as one of the method of the present invention.

[0400] As the “reporter protein”, luciferase derived from firefly or sea pansy, or GFP derived from jerryfish, are preferred.

[0401] As the “reporter gene assay”, methods described below are representative.

[0402] Transgenic cells are generated by transforming cells commonly used in the production of recombinant proteins with expression vector, in which a gene encoding the target protein and a gene encoding the reporter protein are inserted to the vector so that the transcription of the gene encoding the reporter protein to mRNA occurs dependently on the signal of the transcription of the gene of target protein to mRNA. The test substances (described above) are applied to the obtained transformant cells. Analysis that whether the compound affects the expression of transporter molecule can be accomplished by measuring the level of the target protein by indirect measuring of the amount of fluorescence emitted by the reporter protein expressed in parallel with the target protein (for reference, see U.S. Pat. Nos. 5,436,128 and 5,401,629).

[0403] Also, the identification of the compounds using the present assay can be accomplished by manual operation, but it can also be readily and simply done automatically by using so-called High-Throughput Screening using robots (SOSHIKI BAIYO KOUGAKU, 23:521-524; U.S. Pat. No. 5,670,113).

[0404] The “cells” and “substances” used in the methods described above contain the same meaning as defined above.

[0405] The substances identified by the methods of the present invention are very useful as drug for therapy of various diseases considered to be caused by the hyperfunction or deficiency of the AID protein of the present invention or by the deficiency or mutation of the AID gene, or for remission of various symptoms supervene with the diseases.

DESCRIPTION OF DRAWINGS

[0406]FIG. 1 is a is the photograph which shows the production state DNA including an Sa sequence looped out with the class switch recombination in mouse B cell clone CH12F3-2 cultured under the various conditions.

[0407]FIG. 1(a) shows the electrophoretic state of DNA including an Sα sequence looped out with class switch recombination, amplified by PCR using DNA derived from mouse B cell clone CH12F3-2 cultured under the various conditions.

[0408] Lanes 1 and 6 show the electrophoretic state of marker DNAs. Lane 2 shows the electrophoretic state of PCR product using DNA from cells cultured in the condition excluding IL-4, CD40L, TGF β or cycloheximide, as a template. Lane 3 shows the electrophoretic state of DNA product using DNA from cells cultured in the presence of cycloheximide only, as a template. Lane 4 shows the electrophoretic state of PCR product using DNA from cells cultured in the presence of IL-4, CD40L and TGF β, as a template. Lane 5 shows the electrophoretic state of PCR product cultured in the presence of Il-4, CD40L, TGF β, and cycloheximide, as a template.

[0409]FIG. 1(b) shows the result of Southern hybridization for DNA including an Sα sequence looped out with class switch recombination, amplified by PCR using DNA derived from mouse B cell clone CH12F3-2 cultured under the various conditions.

[0410] Lane 1 shows the result of hybridization against PCR product using DNA from cells cultured in the condition excluding any of IL-4, CD40L, TGF β or cycloheximide, as a template. Lane 2 shows the result of Southern hybridization against PCR product using DNA from cells cultured in the presence of cycloheximide only, as a template. Lane 3 shows the result of hybridization against PCR product using DNA from cells cultured in the presence of IL-4, CD40L, and TGF β, as a template. Lane 4 shows the result of hybridization against PCR product using DNA from cells cultured in the presence of IL-4, CD40L, TGF β and cycloheximide, as a template.

[0411]FIG. 2 is a photograph showing the production state of DNA including an Sα sequence looped put with class switch recombination, amplified by PCR using DNA derived from mouse B cell clone CH12F3-2 cultured in various conditions.

[0412]FIG. 2(a) shows the electrophoretic state of DNA from DNA including an Sα sequence looped out with class switch recombination in mouse B cell clone CH12F3-2 cultured in the various conditions, stained with ethidium bromide.

[0413] Lanes 1 and 6 show the electrophoretic state of marker DNAs. Lane 2 shows electrophoretic state of PCR product using DNA from cells cultured in the condition excluding IL-4, CD40L, TGF β or cycloheximide, as a template. Lane 3 shows the electrophoretic state of DNA product using DNA from cells cultured in the presence of cycloheximide only, as a template. Lane 4 shows the electrophoretic state for PCR product using DNA from cells cultured in the presence of IL-4, CD40L and TGF β, as a template. Lane 5 shows the electrophoretic state for PCR product cultured in the presence of Il-4, CD40L, TGF β, and cycloheximide, as a template.

[0414]FIG. 1(b) shows the result of Southern hybridization for DNA including an Sα sequence looped out with class switch recombination, amplified by PCR using DNA derived from mouse B cell clone CH12F3-2 cultured under the various conditions.

[0415] Lane 1 shows the result of hybridization against a PCR product using DNA from cells cultured under the condition excluding any one of IL-4, CD40L, TGF β or cycloheximide, as a template. Lane 2 shows the result of hybridization against a PCR product using DNA from cells cultured in the presence of cycloheximide only, as a template. Lane 3 shows the result of hybridization against a PCR product using the DNA from cells cultured in the presence of IL-4, CD40L, and TGF β, as a template. Lane 4 shows the result of hybridization against a PCR product using DNA from cells cultured in the presence of IL-4, CD40L, TGF β and cycloheximide, as a template.

[0416]FIG. 3 shows the result of Northern blotting using a cDNA fragment coding a radiolabeled 23C9 (AID) protein, against mRNA derived from mouse B cell clone CH12F3-2 cultured under the various conditions.

[0417] Lane 1 shows the result of blotting against mRNA from cells cultured in the condition excluding any one of IL-4, CD40L, or TGF β or cycloheximide. Lane 2 shows the result of blotting against mRNA from cells cultured in the presence of cycloheximide, only. Lane 3 shows the result of blotting against mRNA from cells cultured in the presence of IL-4, CD40L and TGF β. Lane 4 shows the result of blotting against mRNA cultured in the presence of IL-4, CD40L, TGF β, and cycloheximide.

[0418]FIG. 4 shows the result of Northern blotting using radio-labeled cDNA fragment coding 23C9 (AID) protein as a probe against mRNA derived from mouse B cell clone CH12F3-2 cultured in the various conditions.

[0419] Lane 1 shows the result of blotting against mRNA from cells cultured in the condition excluding IL-4, CD40L, TGF β or cycloheximide. Lane 2 shows the result of blotting against mRNA from cells cultured in the presence of cycloheximide, only. Lane 3 shows the result of the blotting against mRNA form cells cultured in the presence of IL-4, CD40L, and TGF β. Lane 4 shows the result of the blotting against mRNA form cells cultured in the presence of IL-4, CD40L, TGF β and cycloheximide.

[0420]FIG. 5 shows the homology between an amino acid sequence of mouse AID protein and that of mouse APOBEC-1

[0421] An amino acid in a closed box shows an identical amino acid. A region in an open box indicates a cytidine deaminase motif. An amino acid with an asterisk (*) or an arrow indicates an amino acid conserved among APOBEC-1 proteins derived from rat, mouse, rabbit, and human.

[0422]FIG. 6 shows a phylogenic tree of various enzymes belonging to a cytosine nucleoside/nucleotide deaminase family, prepared based on cytidine deaminase motif.

[0423]FIG. 7 shows a photograph indicating the electrophoretic state for AID-GST fusion protein in the molecular weight analysis by the gel electrophoresis and silver staining method.

[0424] Lane 1 shows the electrophoretic state for a marker molecule. Lane 2 shows the electrophoretic state for various proteins included in extracts from wild type Escherichia coli DB 5α. Lane 3 shows the electrophoretic state for purified AID-GST fusion protein.

[0425]FIG. 8 shows the electrophoretic state for AID-GST fusion protein by Western blotting using anti-AID protein peptide antibody.

[0426] Lane 1 shows the electrophoretic state for various proteins included in the extract from wild type E. coli DH5α.

[0427] Lane 2 shows the electrophoretic state for purified AID-GST protein.

[0428]FIG. 9 shows a cytidine deaminase activity depending on the concentrations of AID proteins.

[0429]FIG. 10 shows the inhibitory effect of tetrahydrouridine which is an inhibitor specific to cytidine deaminase on a cytidine deaminase activity in AID protein.

[0430]FIG. 11 shows the inhibitory effect of each of 1,10-o-phenanthrolime which is a zinc-chelating agent, and 1,7-o-phenanthroline which is an inactivated isomer thereof on a cytidine deaminase activity in AID protein.

[0431]FIG. 12 is a photograph indicating expression state for mRNA of AID in various tissues in mouse, analyzed by Northern blotting method.

[0432]FIG. 13 is a photograph indicating the expression state for mRNA of AID in various lymphatic tissues in mouse, analyzed by RT-PCR method.

[0433]FIG. 14 is the photograph showing expression state for mRNA of AID as time goes, in activated mouse B cell clone CH12F3-2, analyzed by Northern blotting method.

[0434]FIG. 15 a photograph showing expression state for mRNA of AID in mouse B cell clone CH12F3-2 stimulated with cytokine in various combinations, analyzed by Northern blotting.

[0435]FIG. 16 shows a photograph indicating expression state for mRNA of AID in mouse spleen B cells, stimulated with stimulants in various combinations, analyzed by Northern hybridization method.

[0436]FIG. 17 is a photograph indicating expression state for mRNA of AID in splenocytes derived from mice immunized with sheep red blood cells, analyzed by Northern blotting analysis.

[0437]FIG. 18 shows expression state for mRNA of AID in splenocytes derived from mice immunized with sheep red blood cells, analyzed by RT-PCR.

[0438]FIG. 19 is a photograph indicating localization of expression for AID mRNA in splenocytes derived from a normal mouse or a mouse immunized by sheep red blood cells, specifically, analyzed by in situ hybridization.

[0439] FIGS. 19(A) and (D) indicate the result in the hybridization using a sense AID probe. FIGS. 19(B) and (E) show localization for AID mRNA expression in hybridization using an antisense-AID probe. FIGS. 19(C) and (F) show localization of germinal center in staining test by FITC-labeled PNA. FIGS. 19(A), (B), and (C) indicate the result in the test using spleen tissues derived from normal mouse (before the immunization of sheep red blood cells). FIGS. 19(D), (E), and (F) show the results of the examination using spleen tissue slices prepared 5 days after immunizing a mouse with sheep red blood cells.

[0440]FIG. 20 is a photograph showing the localization of expression for AID mRNA in spleen tissue and payer's patch tissue, respectively, derived from a normal mouse or from a mouse immunized with sheep red blood cells, respectively, analyzed by in situ hybridization.

[0441] FIGS. 20(A), (D), and (G) show the results in the hybridization using a sense AID probe. FIGS. 20(B), (E), and (H) show the localization of the expression for AID mRNA in hybridization using an antisense AID probe. FIGS. 20(C), (F), and (I) show the localization of germinating center in the staining test by FITC-labeled PNA. FIGS. 20(A), (B), and (C) show the result of the examination using spleen tissues derived from a normal mouse (before immunization by sheep red blood cells). FIGS. 20(D), (E), and (F) show the results of the examination using spleen tissue slices prepared 5 days after immunization of a mouse with sheep red blood cells. FIGS. 20(G), (H), and (I) show the results of test using payer's patch prepared 5 days after the immunization of a mouse with sheep red blood cells.

[0442]FIG. 21 schematically shows relative locations of partial nucleotide sequences of human genomic DNA coding human AID protein, which was amplified by PCR using various pairs of primers.

[0443]FIG. 22 schematically shows a degree of homology between an amino acid sequence of mouse AID protein and that of human AID protein. The parts with a closed box are cytidine and deoxycytidylate deaminase zinc-binding region which is an AID protein active region.

[0444]FIG. 23 schematically shows the structure of human genomic DNA including a gene coding human AID protein. One to five shows exon 1, exon 2, exon 3, exon 4, and exon 5, respectively.

[0445]FIG. 24 is a photograph indicating the expression state for human AID mRNA in various types of human tissues, analyzed by RT-PCR.

[0446]FIG. 25 is a photograph indicating a location (localization) of human AID gene on human chromosome, analyzed by Fluorescence in situ hybridization (FISH) method.

[0447] Two points at the tips of arrows show 12p13 where human AID gene exists.

[0448] Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

[0449] The invention is illustrated in details by the following Examples, but not restricted to embodiments described in the Examples.

EXAMPLE 1 Culture of for Mouse B Cell Clone CH12F3-2 and Confirmation of Properties

[0450] Mouse B cell clone CH12F3-2 occurring class switch recombination (CSR) from IgM to IgA, several hours after the stimulation by IL-4, TGF-β, and CD40L, previously isolated by the present inventors, was cultured in the same manner as in the previous report (Immunity, 9:1-10, 1998; Curr. Biol., 8:227-230, 1998; Int. Immunol, 8:193-201, 1996).

[0451] When the CH12F3-2 is stimulated by IL-4, TGF-β, and CD40L, a circular DNA including an S region (switch region) looped out by class switch recombination was detected several hours after the stimulation.

[0452] The following manipulation was conducted according to the previous report (Curr. Biol., 8:227-230, 1998).

[0453] The B cell CH12F3-2 stimulated by IL-4, TGF-β, and CD40L, and that which was not stimulated were cultured for 6 hours in the presence or absence of cycloheximide (200 ng/ml) which is a protein synthesis inhibitor, respectively. Genomic DNA was extracted from each cell, and PCR was conducted with the DNA as a template by following the standard method to amplify circular DNA including an Sμ sequence and an Sα sequence. PCR was conducted by using a pair of primers, αF1 and μR3 and the other PCR was conducted by using a pair of primers, αF1 and μR3.

[0454] As a control, genomic DNA coding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified by PCR.

[0455] PCR product was subjected to the gel electrophoresis by ethidium bromide staining. FIG. 1(a) and FIG. 2 show the results.

[0456] To confirm the presence or absence of the amplification of a circular DNA including the looped-out S region, Southern hybridization was conducted against thee PCR product by using mouse Sa region gene for a hybridization probe, according to the standard method (L. Sambrook E. F., Tom Maniatis., Second edition, Ed. Molecular Cloning (Nolan, C., Ed.) Cold Spring Harbor, 1989). As an Sα gene, a 1,155 bp DNA fragment obtained by digesting 10 kb EcoRI cleaved fragment IgH703 with Hind III and EarI was used (Genbank #D11468, DNA No. 1993-3148) (J, Biol. Chem., Vol. 268, p. 4651-4665). FIGS. 1(b) and 2(b) show the results.

[0457] It has been shown that mouse B cell CH2F3-2 produces the looped-out DNA containing the Sα sequence with the class switch recombination by the stimulation with cytokine, and the production of the DNA is inhibited by the presence of cycloheximide. This result suggested that occurrence of class switch recombination of an immunoglobulin gene needs a novel synthesis of a protein in the very early stage after the stimulation and the protein is deeply involved in the induction of the class switch.

EXAMPLE 2 Identification of a Gene which Expression is Improved in Mouse B Cell CH12F3-2 Stimulated by Cytokine

[0458] A gene which is presumably expressed in the early stage after mouse B cell clone CH12F3-2 is stimulated, and presumably play a role of introducing class switch recombination of an immunoglobulin gene was attempted to be isolate from the CH12F3-2 cells by the suppression subtract hybridization (SSH) (Proc. Natl. Acad. Sci. USA, 93:6025-6030, 1996; Anal. Biochem., 240:90-97, 1996) using the inhibitory PCR effect (Nucleic Acids Res., 23:1087-1088, 1995).

[0459] A cDNA library necessary for subtraction cloning was prepared by using PCR-Select Subtraction Kit (CLONTECH, Catalogue NO: K1804-1) by following the instruction manual supplemented with the kit in the experimental manipulation.

[0460] PolyA⁺RNA was isolated from each of mouse B cell clone CH12F3-2 stimulated with IL-4, TGF-β and CD40L for 5 hours, the same cells stimulated with the cytokines for 12 hours, and the cells which were not stimulated, by following the reported method (Nucleic Acids Res., 26:911-918, 1998) and treated with DNaseI to eliminate genomic DNA which may be mixed. Then cDNA was prepared based on each polyA⁺RNA sample using reverse transcriptase according to the standard method. Each cDNA prepared from mouse B cell clone CH12F3-2, treated with the above cytokines for 5 or 12 hours was mixed with same mole amounts to be used as a tester cDNA. On the other hand, cDNA derived from unstimulated cells was used as a driver cDNA.

[0461] Subtraction was conducted by adding the driver cDNA into the tester cDNA according to the above previous report and the experimental manipulation manual. The efficiency of subtraction was monitored by adding a small amount (1:1000 mole ratio) of Φ X174 phage DNA cleaved at the restriction enzyme site Hae III, as a control, into the tester cDNA. After the subtraction, the phage DNA was concentrated to a mole ratio of about 100 times.

[0462] The subtracted cDNA was cloned to T-vector (Promega) according to the standard method to prepare a plasmid library. In the same manner as in the previous report, 2000 colonies in the library were screened by the differential hybridization method (Nucleic Acids Res., 26:911-918, 1998; Medical immunity, 29:17, p. 451-459, 1997). Each of the above tester cDNA and driver cDNA was radiolabeled to be used for hybridization. Clones including Φ X174 phage DNA were selected by hybridizing Φ X174 phage DNA with a replicant filter.

[0463] One hundred fifteen clones emitting a stronger signal than the radio-labeled driver cDNA probe against radio-labeled tester cDNA probe were identified and a nucleotide sequence of each clone was determined by using a DNA sequencer.

[0464] Northern blotting was conducted against mRNA obtained from mouse B cell clone CH12F3-2 stimulated with IL-4, TGF-β and CD40L or the same lines unstimulated, using the radio-labeled DNA inserted into the each clone as a probe, according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition, Ed. Molecular Cloning (Nolan, C., Ed.), Cold Spring Harbour, 1989). As a result, the enhanced expression corresponding to the stimulation with the above cytokines was observed in 23 among 115 clones. Gene fragments coding 7 different types of proteins, including genes coding the 3 kinds of known proteins and 4 kinds of novel proteins were found to be inserted into the 23 clones. Specifically, the expression of the 7 kinds of genes were found to be enhanced in mouse B cell clone CH12F3-2 by the stimulation with IL-4, TGF-β and CD40L.

[0465] <The Known Proteins>

[0466] ABCD-1/MDC (8 clones)

[0467] IFNg receptor (2 clones)

[0468] I-a (MHC class II) (1 clone)

[0469] <Novel Proteins>

[0470] 23C9 (3 clones)

[0471] 15B11 (7 clones)

[0472] 8B9 (1 clone)

[0473] 16A9 (1 clone

[0474] As it has been known that the expression of the above I-a gene and ABCD/MDC gene is enhanced by stimulating mouse spleen B cell with IL-4 and CD40L, it was confirmed that the subtraction cloning was effectively conducted (J. Exp. Med., 188:451-463, 1998; Immunity, 5:319-330, 1996)

EXAMPLE 3 Expression of mRNA for a Novel Protein 23C9 in Mouse B Cell Clone CH12F3-2

[0475] The degree of enhanced expression of gene coding a novel protein 23C9 in mouse B cell clone CH12F3-2 stimulated with IL-4, TGF-β and CD40L was analyzed according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition, Ed. Molecular Cloning (Nolan, C., Ed.), Cold Spring Harbour, 1989) by Northern blotting.

[0476] Mouse B cell clone CH12F3-2 was cultured in the presence of one of the following regents for 12 hours.

[0477] (1) IL-4, TGF-β and CD40L only.

[0478] (2) Cycloheximide which is a protein synthesis inhibitor (200 ng/ml), only

[0479] (3) IL-4, TGF-β and CD40L as well as Cycloheximide (200 ng/ml)

[0480] Northern blotting was conducted against mRNA (10 μg for each group) obtained in the same manner as the previous report (Nucleic Acid Res., 26:911-918, 1998) from each group of treated cells using a radio-labeled cDNA fragment (1,020 bp)coding a novel protein 23C9, obtained in the above Example, according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition, Ed. Molecular Cloning (Nolan, C., Ed.), Cold Spring Harbour, 1989).

[0481] As a control examination, Northern blotting was conducted for mRNA derived from B cell clone CH12F3-2 cultured without any one of the above cytokines, or cycloheximide.

[0482] The amount of mRNA to be electrophoresed was adjusted using the amount of mRNA in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an index. DNA amplified by RT-PCR using GF primer and GR primer was used as a probye for blotting of GAPDH mRNA (Location of nucleotides: 566-1016, Genbank U5299) (Immunity, 9:1-10, 1998).

[0483]FIGS. 3 and 4 show the results.

[0484] The expression of mRNA for a novel protein 23C9 was extremely strong in mouse B cell clone CH12F3-2 stimulated with IL-4, TGF-β and CD40L, while the expression in unstimulated cells was extremely weak. Expression of the mRNA in the stimulated cells was inhibited by the presence of a protein synthesis inhibitor. Moreover, in the stimulated cells, two bands indicating the expression of mRNA comprising different nucleotide lengths were detected.

[0485] Expression of mRNA for a novel protein 23C9 in each of the following mouse cell lines which do not originally comprise class switch recombination was examined by Northern blotting in the same manner as in the above.

[0486] B cell lines (1yD9, BA/F3, 70Z/3, WEHI231), T cell lines (EL-4, 2B4), myeloma cell lines (X63, HEHI-3). Fibroblast lines (L929, NIH3T3,) the other cell lines (F2, P815, ST2).

[0487] The expression of mRNA for the novel protein 23C9 was not observed in any cells.

EXAMPLE 4 Cloning of a Full Length cDNA Coding a Novel Protein 23C9

[0488] Four different positive clones were obtained by screening cDNA library (Nucleic Acids Res., 26:911-918, 1998) prepared from mouse B cell clone CH12F3-2 stimulated with IL-4, TGF-β, and CD40L, using a cDNA fragment (1,020 bp) coding the novel protein 23C9, obtained in the above Example as a probe. A nucleotide sequence of each clone was determined by using a DNA sequencer according to the standard method.

[0489] One clone comprises a 1.2 kb nucleotide length and a single reading frame (OPF) with 1 polyadenylation site. The other 3 clone comprise a 2.4 kb nucleotide length and 2 polyadenylation sites. A nucleotide sequence in the 1.2 kb part at the 5′ side in the latter clones was identical to that of the nucleotides in the 1.2 kb DNA in the former (SEQ ID NO:1).

[0490] Two different mRNA transcripts detected in Northern blotting in the above Example (FIGS. 3 and 4) were predicted to correspond to transcripts for each of the above 1.2 kb and 2.4 kb, transcribed using the polyA site at 3′ end and the polyA site at the 5′ end.

[0491] A cDNA fragment coding the novel protein 23C9 used as a probe in the above (1,020 bp) was found to be a nucleotide sequence of from 847 to 1866 in the full length cDNA of 23C9.

[0492] A nucleotide sequence near a first initiation codon in each cDNA was fit to Kozak's rule (Nucleic Acids Res., 15:8125-8148, 1987). In the 2.4 kb cDNA, ATTTA which is a motif capable of mediating quick degradation of mRNA (Blood, 83:3182-3187, 1994) was present in the 2 sites in the untranslational region in the 3′side.

[0493] An open reading frame (ORF) of cDNA coding the novel protein 23C9 consisted of 198 amino acids with the expected molecular weight of about 24 kDa (SEQ ID NO:2). As a result of homology searching with known proteins by database, an amino acid sequence of ORF of the novel protein 23C9 comprised 34% amino acid homology with apolipoprotein B mRNA editing enzyme, catalytic polypeptide-1 (APOBEC-1) (Science, 260:1816-1819, 1993; J. Biol. Chem., 268:20709-20712. 1993). GenBank and EMBL were used as DNA data base. SwissPlot was used as protein database. BLAST program (J. Mol. Biol., 215:403-410, 1990) and FASTA program (Proc. Natl. Acad. Sci. USA., 85:2444-2448 1988) were used for database search.

[0494]FIG. 5 shows an amino acid sequence of ORF of the novel protein 23C9 and an alignment between the sequence and that of mouse APOBEC-1 amino acid sequence.

[0495] As a result of motif search on online using PROSITE (Nucleic Acids Res., 11:2013-2018, 1992), the APOBEC-1 like novel protein 23C9 comprises cytidine/deoxycytidine deaminase motif which is conserved in a amino acid sequence of a protein belonging to cytosine nucleoside/nucleotide deaminase family which constructs a large family and is an activation site of a deaminase activity. A cytosine nucleoside/nucleotide deaminase family is classified into RNA editing deaminase, cytidine/deoxycytidylate deaminase, and CMP/dCMP deaminase based on the substrate specificity and homology in the activation sites (Cell, 81:187-195, 1995).

[0496] A phylogenic tree was prepared based on the alignment among regions in APOBEC-1 which is an RNA editing deaminase, cytosine nucleoside deaminase, cytosine nucleotide deaminase, and cytidine deaminase motif in the novel protein 23C9. The sequences in the known proteins used for the comparison were obtained from GenBank, as follows.

[0497] Human derived nucleoside deaminase: L27943

[0498] Mouse derived nucleoside deaminase: AA388666

[0499]S. subtilis derived nucleoside deaminase: U18532

[0500]E. coli derived cytidine deaminase: X63144

[0501] Rabbit derived APOBEC-1: U10695

[0502] Human derived APOBEC-1: L25877

[0503] Rat derived APOBEC-1: U10695

[0504] Mouse derived APOBEC-1: U21951

[0505] T2/T4 phage derived nucleotide deaminase: J05172

[0506] Human derived nucleotide deaminase: L12136

[0507]S. cerevisiae derived nucleotide deaminase: U10397

[0508]FIG. 6 shows the result. Cytidine deaminase motif in the novel protein 23C9 was rather relative to a subgroup of RNA editing deaminase than subgroups of nucleoside deaminase and nucleotide deaminase.

[0509] On the other hand, a leucine-rich region existing at the C-terminus of APOBEC-1 is thought to be important for protein-protein interaction (Proc. Natl. Acad. Sci. USA., 91:8522-8526, 1994; J. Biol. Chem., 269:21725-21734, 1994). The novel protein 23C9 also comprised a leucine-rich region at the C-terminus. Four leucines in the region o 23C9 were conserved in the leucine rich regions of APOBEC-1 in rabbit, rat, mouse and human.

[0510] It has been known that Phe66, Phe87, His61, Glu63 and Cys93 are essential for binding of APOBEC-1 to RNA, and all these amino acid residues were conserved in the primary structure of 23C9 (Trends Genet., 12:418-424, 1996; Cell, 81:187-195, 1995; J. Biol. Chem., 270:14768-14775 1995; J. Biol. Chem., 270:14762-14767, 1995). From this fact, 23C9 protein is predicted to comprise an RNA editing deaminase activity.

[0511] Moreover, cytidine deaminase derived from APOBEC-1 and E. coli (ECCDA) are known to comprise a pseudoactive site domain at the C-terminus and the 23C9 protein also comprised a pseudoactive site domain same as in the APOBEC-1. This indicates that 23C9 protein is more relative to APOBEC-1 and ECCDA than deaminase proteins in the other groups.

[0512] From these facts, the novel protein 23C9 was named activation-induced cytidine deaminase (AID). The novel protein 23C9 was called AID hereafter.

EXAMPLE 5 Preparation of the AID-GST Fusion Protein

[0513] The cDNA coding a full length AID cloned in the above Example was amplified by PCR with a pair of primers, AID-138 (SEQ ID NO:3) and AID-161 (SEQ ID NO:4), a pair of primers, AID-118 (SEQ ID NO:5) and AID-119 (SEQ ID NO:6) and Taq Polymerase by following the standard method. As there is an intron between AID-118 and AID-119, a PCR product derived from AID genomic DNA can be easily distinguished.

[0514] The obtained PCR product was subcloned to pGEX4T1 vector (Pharmacia) according to the standard method. A nucleotide sequence of the vector was determined and the absence of point mutation derived from the use of Taq polymerase in the full length AID cDNA cloned to the vector was confirmed.

[0515]E. coli DH5α was transformed with the vector according to the standard method. The obtained transformants were cultured, and a full length AID cDNA was expressed as a fusion protein with glutathione S-transferase (GST). The AID-GST fusion protein was extracted in the same manner as in the previous report, and purified using glutathione agarose affinity chromatography (J. Biol. Chem., 270:14768-14775 1995).

[0516] A molecular weight of the purified AID-GST fusion protein was analyzed by following the standard method using 10% SDS-PAGE and silver staining. A protein extracted from wild type E. coli DH5a was used as a control. FIG. 7 shows the result.

[0517] As expected, the fusion protein was detected as a band comprising a molecular weight of about 49 kDa. Minor bands detected under the about 49 kDa were thought to be decomposed proteins, frequently generated in the purification process in general.

[0518] A molecular weight of the purified AID-GST fusion protein was analyzed by the Western blotting method according to the standard method (Genomics, 54:89-98, 1998). Anti-AID protein antibody to be used for the assay was prepared by immunizing a commercial rabbit for the experiment with multiple antigen peptides including synthetic peptides corresponding to amino acid NO: 116 to 132 of the AID protein of the present invention (Proc. Natl. Acad. Sci. USA., 85:5409, 1988).

[0519]FIG. 8 shows the result.

EXAMPLE 6 Cytidine Deaminase Activity of the AID Protein

[0520] A cytidine deaminase activity of AID was measured by the same method as in the previous report (J Biol. Chem. 270:14768-14775, 1995).

[0521] The purified AID-GST fusion protein prepared in the above (2, 4, 6, 8, 10, 20, 40, 60, 100, 200, 300, 400, and 600 ng) was incubated in the buffer (pH 7.5, the total amount 10 μl) containing 45 mM Tris with 3.3 μCi [³H] deoxycytidine (24.8 Ci/mmol, DuPont) and 250 μM cytidine for 2 to 4 hours. The reaction was terminated by adding deoxycytidine (2 μl of 10 μg/ml) and deoxyuridine (2 μl of 10 μg/ml). Insoluble substances were removed by centrifugation, and the reaction mixture (4 μl) was subjected to the polyethylene imine-cellulose thin layer chromatography plate (VWR). The plate was developed in isopropyl alcohol/10% HCl (7:2 v/v). The plate was exposed to ultraviolet light (254 nm) for visualization and bands corresponding to deoxycytidine and deoxyuridine were collected, added to Ultima Gold scintillation solution to be quantified by liquid scintillation photometer (Packard)

[0522]FIG. 9 shows the result. As a result, AID protein showed the cytidine deaminase activity depending on the concentrations.

[0523] An inhibitory effect of tetrahydrouridine (THU; 0 to 40 μM) (Calbiochem, USA) which is an inhibitor specific to cytidine deaminase, on the cytidine deaminase activity in the AID-GST fusion protein (300 ng) was measured by the same method described above.

[0524]FIG. 10 shows the result. The cytidine deaminase activity of AID protein was inhibited dependently on the concentrations of THU.

[0525] Each inhibitory effect of 1,10-o-phenanthroline (0 to 20 mM) which is a zinc-chelating agent and its inactive form isomer 1,7-o-phenanthroline (0 to 20 mM) on the cytidine deaminase activity in the AID-GST fusion protein was measured in the same manner as described below.

[0526]FIG. 11 shows the result. The cytidine deaminase activity of AID protein was inhibited by 20 mM 1,10-o-phenanthroline by about 91%. 1,7-o-phenanthroline which is the inactive isomer only inhibited about 13%. These results indicate that AID protein is a zinc-dependent cytidine deaminase, same as APOBC-1.

EXAMPLE 7 Avidity of AID Protein with AU-rich RNA

[0527] A recombinant APOBEC-1 binds to Au-rich RNA (Trends Genet., 12:418-424, 1996; Cell, 81:187-195, 1995; J. Biol. Chem., 270:14768-14775, 1995; J. Biol. (Chem., 270:14762-14767, 1995), and progresses RNA editing for apoB in the presence of chicken extract including co-factor.

[0528] Since the AID protein has a functional cytidine deaminase activity as well as a structural similarity with APOBEC-1, to examine an RNA editing activity in the AID protein, avidity to AU-rich RNA (5-AU) and apoB RNA which are RNA substrate for APOBEC-1 was examined.

[0529] The AID protein did not show avidity to AU-rich RNA (5-AU) in the gel retardation assay. In in vitro apoB RNA assay, conversion from cytidine (C) to uridine (U) was not observed.

EXAMPLE 8 Expression Distribution of AID mRNA in Tissues

[0530] The expression of AID mRNA in each tissue was examined by Northern blotting according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition, Ed. Molecular Cloning (Nolan C., Ed.), Cold Spring Harbour, 1989; Experimental Medicine, Suppl., “Genetic Engineering Hand Book”, published by Yodosha, p. 133-140, 1992).

[0531] PolyA⁺RNA (2 μg each) obtained from cells derived from each tissue in mice (muscle, spleen, lung, heart, lymph node, brain, kidney, thymus, testis, liver) according to the previous report (Nucleic Acids Res., 26:911-918, 1998) was used as a sample. Radiolabeled cDNA fragment (1,020 bp) coding AID (23C9) obtained in the previous Examples was used as a probe for blotting polyA⁺RNA.

[0532] As a control, mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was blotted in the same manner. AS a probe for blotting GAPDH mRNA, DNA amplified by PCR using GP primer and GR primer was used. (Nucleotide location: 566-1016, Genbank, U52599) (Immunity, 9:1-10, 1988).

[0533]FIG. 12 shows the result.

[0534] As a result, AID mRNA was strongly expressed in mesenteric lymph node. In addition, weak expression was observed in spleen.

EXAMPLE 9 Expression of AID mRNA in Various Lymphatic Tissues

[0535] The expression of AID mRNA in each lymphatic tissue was analyzed by RT-PCR according to the standard method (Immunity, 9:1-10, 1998).

[0536] cDNA was prepared according to the standard method using polyA⁺RNA obtained from cells derived from various lymphatic tissues (Payer's patch, mesentric lymph node, axillary lymph node, spleen, bone marrow, thymus) in the same manner as in the previous report (Nucleic Acids Res., 26:911-918, 1998), for mRNA as a sample, as a template. AID cDNA and GAPDH cDNA were amplified using the obtained cDNA as a template. The pair of primers, AID-138 (SEQ ID NO:3) and AID-161 (SEQ ID NO. 4) in the above, a pair of primers AID-118 (SEQ ID NO:5) and AID-119 (SEQ ID NO:6) and Taq polymerase were used for PCR of AID cDNA. As there is an intron between AID-118 and AID-119, a PCR product derived from the AID genomic DNA sequence can be easily distinguished.

[0537]FIG. 13 shows the result.

[0538] AID cDNA was detected in all lymphatic tissues except for thymus. In particular, the obvious expression was observed in peripheral lymphatic organs, such as lymph node or Payer's patch. On the other hand, the expression in primary lymphatic organs was weak in comparison with that in the peripheral lymphatic organs.

EXAMPLE 10 Expression of AID mRNA as Time Goes in Activated Mouse B Cell Clone CH12F3-2

[0539] Expression of AID mRNA as time goes in activated mouse B cell clone CH12F3-2 stimulated with IL-4, TGF-β, and CD40L for 0 to 60 hours was analyzed by Northern blotting according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition Ed. Molecular Cloning (Nolan, C., Ed.). Cold Spring Harbor, 1989).

[0540] Mouse B cell clone CH12F3-2 was cultured in the presence of IL-4, TGF-β, and CD40L for the various periods (0, 3, 5, 12, 24, 36 48 or 60 hours).

[0541] Northern blotting was conducted against mRNA (10 μg in each group) obtained from each culture group in the same manner as in the previous report (Nucleic Acids Res., 26:911-918, 1998) using a radiolabeled cDNA fragment coding AID (23C9) obtained in the previous Examples, as a probe, according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition Ed. Molecular Cloning (Nolan, C., Ed.). Cold Spring Harbor, 1989).

[0542] The amount of mRNA to be gel-electrophoresed was adjusted by using mRNA of GAPDH as an index. DNA amplified by RT-PCR using GF primer and GR primer was used as a probe for blotting GAPDH mRNA (Nucleotide location: 566-1016, Genbank U52599) (Immunity, 9:1-10, 1998).

[0543]FIG. 14 shows the result.

[0544] It was shown that The expression of AID mRNA in mouse B cell clone CH12F3-2 was too small to be detected without the stimulation by cytokines, but that the expression was initiated 3 hours after the stimulation by cytokines (described in the above), was maximum 12 hours after the stimulation (more than about 15 times), and was gradually decreased from 48 hours after the stimulation.

EXAMPLE 11 Cytokine Specificity to Inducing Expression of AID mRNA in Mouse B Cell Clone CH12F3-2

[0545] Cytokine specificity to inducing expression of AID mRNA in mouse B cell clone CH12F3-2 was analyzed by Northern blotting according to the standard method L. Sambrook, E. F., Tom Maniatis., Second edition Ed. Molecular Cloning (Nolan, C., Ed.), Cold Spring Harbour, 1989).

[0546] Mouse B cell clone CH12F3-2 was cultured in the presence of various combinations of cytokines (one or more selected from IL-4 TGF-β, CD40-L) for 12 hours. Northern blotting was conducted against mRNA (10 μg in each group) obtained from each culture group in the same manner as in the previous report (Nucleic Acids Res., 26:911-918, 1998) using a radio-labeled cDNA fragment (1,020 bp) coding AID (23C9) obtained in the previous Example, according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition Ed. Molecular Cloning (Nolan, C., Ed.). Cold Spring Harbor, 1989).

[0547] The amount of mRNA to be gel-electrophoresed was adjusted by using mRNA of GAPDH as an index. DNA amplified by RT-PCR using GF primer and GR primer was used as a probe for blotting GAPDH mRNA (Nucleotide location: 566-1016, Genbank U52599) (Immunity, 9:1-10, 1998).

[0548]FIG. 15 shows the result.

[0549] Expression induction of AID-mRNA was small by solely any one kind of cytokines. On the other hand, when 3 kinds of cytokines described above were used at the same time, the maximum expression induction of AID-mRNA was observed.

[0550] As described in the above Example 3, because expression induction of AID mRNA was inhibited by cycloheximide which is an inhibitor for protein synthesis, it is hypothesized that enhanced expression of AID mRNA needs de novo synthesis.

EXAMPLE 12 Expression Induction of AID mRNA in Spleen B Cell by Stimulation

[0551] The presence or absence of expression induction of AID mRNA by stimulation which may activate B cell and induce class switch recombination of immunoglobulin was examined.

[0552] Spleen B cell was purified and obtained from BALB/c mouse (6 to 12-week old, Shimizu Experimental Materials (SLC)) according to the standard method. Dead cells and cell fragments were removed by Ficoll density gradient centrifugation after the process of removing T cells. The purified spleen B cell was cultured for 4 days in the presence of a stimulus in various combinations (one or more selected from IL-4, TGF-β, CD40L and LPS (lipopolysaccharide) in the same manner as in the previous report (Nucleic Acids Res., 26:911-918, 1998). LPS derived from Salmonella typhosa (50 μg/ml, Sigma) was used.

[0553] Northern blotting was conducted against mRNA (15 μg in each group) obtained from each culture group in the same manner as in the previous report (Nucleic Acids Res., 26:911-918, 1998) using a radio-labeled cDNA fragment coding AID (23C9) obtained in the previous Example, according to the standard method (L. Sambrook, E. F., Tom Maniatis., Second edition Ed. Molecular Cloning (Nolan, C., Ed.). Cold Spring Harbor, 1989).

[0554] The amount of mRNA to be gel-electrophoresed was adjusted by using mRNA of GAPDH and 28S ribosomal RNA as an index. DNA amplified by RT-PCR using GF primer and GR primer was used as a probe for blotting GAPDH mRNA (Nucleotide location: 566-1016, Genbank U52599) (Immunity, 9:1-10, 1998).

[0555]FIG. 16 shows the result.

[0556] The enhanced expression of AID mRNA by the stimulation with LPS only or LPS+IL-4, or LPS+TGF-β was observed in normal mouse spleen B cells.

EXAMPLE 13 Induced Expression of AID mRNA in Vivo

[0557] It was examined whether the expression induction of AID mRNA by various stimulation in vitro would occur in vivo.

[0558] BALB/c mouse (6 to 12-week old, five individuals in each group, SLC) was immunized by intraperitoneally administering sheep red blood cell (SRBC) (1×10⁸ cells, Cosmo Bio.). In the living body immunized by SRBC, it has been known that clonal expansion and germinal center formation occur after immunoresponse, and class switch recombination of an immunoglobulin gene and affinity maturation are caused.

[0559] PolyA⁺RNA was prepared from splenocytes isolated from spleen excised from each individual before (day 0) and after (day 2, 5 and 13) the immunization.

[0560] The PolyA⁺RNA (2 μg each) was subjected to the Northern blotting using the radiolabeled cDNA fragment (1,020 bp) coding AID (23C9) as a probe in the same manner as the above Examples. The amount of mRNA to be gel-electrophoresed was adjusted using mRNA of GAPDH as an index in the same manner as in the above Examples.

[0561]FIG. 17 shows the result.

[0562] The minimum amount of expressed AID mRNA was detected before immunization of SRBC (day 0), however, a significant enhancement of expression (about 4 to 5 times) was observed day 5 and day 13 after the immunization.

[0563] Moreover, to analyze in which cell type enhanced expression of AID mRNA occurs, RT-PCR was conducted by the standard method (Immunity, 9:1-10, 1998).

[0564] Red blood cells were removed from splenocytes obtained from spleen which was obtained 5 days after the immunization of SRBC in the same manner as the above, and T cells and non-T cells were separated using nylon fiber (Wako Pure Chemicals) in the same manner as in the previous report (Eur. J. Immunol., 3:645-649, 1973). T cell fraction contained more than 90% of CD3 positive cells, and less than 20% B 220 positive cells. T-cell fraction (removal of B cells) and B-cell fraction were concentrated by MACS method with magnetic beads conjugated to anti-CD19 antibody (Miltenyi Biotech.). B220 positive B cells included in the fraction in which T cells were removed were 5% or less. On the other hand, B220 positive B cells included in the fraction in which CD19 positive cells were concentrated were 60% or more.

[0565] cDNA was prepared by reverse transcriptase according to the standard method using polyA⁺RNA prepared from each fractionated cell group. AID cDNA and GAPDH cDNA were amplified by PCR using the obtained cDNA as a template. For PCR of AID cDNA, the previously described pair of primers, AID-138 (SEQ ID NO:3) and AID-161 (SEQ ID NO:4), and the previously described pair of primers, AID-118 (SEQ ID NO:5) and AID-119 (SEQ ID NO:6), as well as Taq polymerase were used.

[0566]FIG. 18 shows the result.

[0567] As a result, in the CD19 positive B cell fraction and non-T cell friction, amplification of AID cDNA was observed. Specifically, it was demonstrated that enhanced expression of AID mRNA induced by immunization by SRBC occurs in spleen CD19 positive B cells.

EXAMPLE 14 Localization of AID mRNA Expression in Lymphatic Organs

[0568] It was found that timing of enhanced expression of AID mRNA in spleen almost consistent with the initiation of germinal center (GC) formation after immunization of SRBC, from the result of the previous Examples. In this examination, an precise localization of AID mRNA expression in lymphatic organs was analyzed using in situ hybridization.

[0569] AID cDNA cleaved out by digesting pGEX4T1 vector in which cDNA coding AID protein has been subcloned, with EcoRI and XhoI was subcloned into plasmid pBluescriptSK (+) (Stratagene). The plasmid was digested with EcoRI or XhoI to obtain linearized plasmid DNA and transcribed into RNA using the plasmid as a template in the presence of digoxigenin-labeled rUTP (Boehringer-Mannheim) using T3 RNA polymerase or T7 RNA polymerase to prepare each of digoxigenin-labeled antisense probe and sense probe.

[0570] On the other hand, frozen tissue slices were prepared by immobilizing with paraformaldehyde from each of spleen and payer's patch in a normal mouse as a lymphatic organ sample. A normal mouse was immunized with SRBC in the same manner as in the above Examples, and frozen tissue slices immobilized with paraformaldehyde from spleen obtained 5 day after the immunization.

[0571] Hybridization was conducted by applying the digoxigenin-labeled antisense AID probe or sense AID probe to each of the slides furnished with each of immobilized slices. Hybridized digoxigenin-labeled AID probe was detected using anti-digoxigenin antibody conjugated with alkaline phosphatase. The localization of anti-digoxigenin antibody conjugated to digoxigenin on the probe was identified by detecting a phosphatase reactant (dark purple color). This analysis was conducted using a light transmission microscope.

[0572] In situ hybridization and detection of riboprobe in this examination were conducted in the same manner as in the previous report (J. Comp. Neurol., 333:398-416, 1993).

[0573] The location of germinal center in each tissue slice was identified by staining with PNA (Vector) conjugate with FITC and observing with a inflorescent microscope.

[0574]FIGS. 19 and 20 show the result.

[0575] In the examination using the antisense AID probe, multiple obvious focal signals were observed in spleen tissue slices derived from SRBC immunized mouse (day 5 after the immunization) (FIGS. 19(E) and 20(E)), however, any signals were not detected in spleen tissue slices derived from mice which were not immunized with SRBC (FIGS. 19(B) and 20(B)). This result is consistent with the result of Northern blotting obtained in the above Example (FIG. 17). Existence of germinal center was observed both in spleen tissue slices derived from SRBC-immunized mouse (day 5 after the immunization) (FIGS. 19(F) and 20(F)) as well as in the normal payer's patch (FIG. 20(I)), by staining with FITC-labeled PNA. The expression of AID mRNA was found to localize in germinal center in the both tissue slices.

[0576] In the examination using sense AID probe, any signals as background were not detected in tissue slices of spleen or of payer's patch regardless of presence or absence of the immunization by SRBC.

[0577] This result indicates that expression induction of AID mRNA occurs in specifically to germinal center B cells, activated by stimulation with an antigen.

EXAMPLE 15 Isolation of Genomic DNA Coding AID Protein Derived from Human

[0578] 15-1 Preparation of Probes for Hybridization

[0579] PCR was conducted using an expression vector prepared by inserting cDNA coding a full length mouse AID protein, prepared in Example 5 into a plasmid vector pGEX4T1, as a template with a pair of primers (Primer 170: SEQ ID NO:16 and primer 181: SEQ ID NO: 179, according to the standard method described in the above.

[0580] The obtained PCR product was purified by the standard method described above and a nucleotide sequence of the purified DNA was determined by the direct sequence method to confirm that the purified DNA is the nucleotide sequence coding a full length of mouse AID protein. This purified DNA was used as a probe for hybridization in the following experiments.

[0581] 15-2 Screening of Human Genomic DNA Library

[0582] The probe prepared in the above was labeled in the same manner as for the radioactive probe in the above Northern hybridization to make a probe radio-labeled by a radioactive isomer.

[0583] Using the labeled probe, a commercial human genomic DNA library (catalogue No. HL1067j; Lot No. 45003; CLONTECH) was screened by the cross hybridization according the standard method.

[0584] Washing after the hybridization was conducted twice in 2×SSC (including 0.1% SDS, under the room temperature, 10 min), and twice in 2×SSC (including 0.1% SDS, 65° C., 30 min). Phage DNA was subcloned by purifying phage DNA and inserting about 22 kb genomic DNA obtained by cleaving at NotI restriction enzyme site in the phage DNA, into Not I restriction enzyme site in plasmid pZero-2.1. This plasmid was named 3CpZero.

[0585] A DNA fragment obtained by digesting 3CPZero with PstI was ligated to the PstI site of plasmid pBlueScript KS (Toyobo) and E. coli was transformed with this ligated DNA.

[0586] Transformants were screened by the colony hybridization using the labeled probe prepared in the above according to the standard method, and multiple positive clones were obtained.

[0587] A nucleotide sequence of human genomic DNA inserted into each positive clone was analyzed and multiple clones containing genomic DNA of DNA coding human AID protein were identified.

[0588] Among the multiple clones, nucleotide sequences of genomic DNA containing DNA coding human AID protein contained in two clones were described in SEQ ID NOs:9 and 10, respectively.

[0589] Moreover, a nucleotide sequence of genomic DNA including DNA coding human AID protein included in the positive other clone was shown in SEQ ID NO:35.

EXAMPLE 16 Isolation of cDNA Coding a Full-length Human AID Protein and Preparation of Human AID Protein

[0590] By comparing a nucleotide sequence of genomic DNA including a coding region of the obtained human AID protein with cDNA nucleotide sequence coding a full-length mouse AID protein determined in the above, a human AID protein coding region in the human genomic DNA was deduced.

[0591] A pair of primers for RACE-PCR was designed based on the deduced nucleotide sequence of the coding region in the human AID protein (Primer 22: SEQ ID NO:18, and primer 25: SEQ ID NO:19).

[0592] RACE-PCR was conducted using mRNA prepared from human B Lymphoma cell line RAMOS as a template with the above pair of primers according to the previous report (J. Biol. Chem., 274:18470-18476, 1999) by following the standard method. A nucleotide sequence of the obtained PCR product was determined and cDNA coding a full length human AID protein was obtained (cDNA sequence: SEQ ID NO:7, and amino acid sequence: SEQ ID NO:8).

[0593] As a result, human AID protein (SEQ ID NO:8) has extremely high homology in amino acid sequences with a mouse AID protein (SEQ ID NO:2) (FIG. 22). Amino acid sequences in Cytidine and deoxycytidilate deaminase zinc-binding region which is an active region in AID protein (both mouse AID and human AID SEQ ID NO:56 to 94) were completely consistent (conserved) between mouse and human.

[0594] As a partial amino acid sequence (amino acid NO: 116 to 132 in SEQ ID NO:2) of mouse AID protein used for the preparation of anti-AID protein antibody (Example 5) was completely consistent with a corresponding amino acid sequence (amino acid NO: 116 to 132 in SEQ ID NO:8) of human AID protein, the anti-AID protein antibody was expected to comprise cross-reactivity not only with mouse AID protein but also with human AID protein.

[0595] Human AID cDNA obtained in the above was reconstructed according to the standard method in the manner of genetic engineering so that His-AID fusion protein added with His-tag (a peptide of histidine repeated 10 times) at N-terminal in the human AID protein was produced, and an expression vector was prepared by inserting the cDNA into a plasmid pEF-BOS (Unexamined published Japanese patent No. Hei 2-242687). The vector was introduced into monkey kidney derived cell line COS7 by lipofection using LIPOFECTAMINE (GIBCO BRL) according to the standard method. The obtained transgenic cells were cultured by the standard method and His-human AID fusion protein was transiently expressed. His-human AID fusion protein was extracted and purified in the same method as the previous report, and the production of His-human AID fusion protein was analyzed by Western blotting with the anti-AID antibody prepared in Example 5 and a commercial anti-His tag antibody according to the standard method. As a result, the His-AID protein was detected as a band comprising about 31 kDa molecular weight in all cases using any antibody.

EXAMPLE 17 Determination of Exons in Genomic DNA Coding Human AID Protein

[0596] Based on the information for the nucleotide sequence o cDNA coding the full length human AID protein above, exons in the nucleotide sequences for genomic DNA coding human AID protein in the above were determined.

[0597] As a result, it was confirmed to consist of 5 exons.

[0598] Exon 1 (Nucleotide sequence: SEQ ID NO:11);

[0599] Exon 2 (Nucleotide sequence: SEQ ID NO:12);

[0600] Exon 3 (Nucleotide sequence: SEQ ID NO:13);

[0601] Exon 4 (Nucleotide sequence: SEQ ID NO:14); and

[0602] Exon 5 (Nucleotide sequence: SEQ ID NO:15).

[0603] The exon 1 contains a translation initiation codon ATG which codes the first methomine (Amino acid No: 1 of SEQ ID NO:8) in human AID protein, and the initiation codon corresponds to nucleotide NOs: 80 to 82 in SEQ NO: 11.

[0604] Specifically, the genomic DNA including DNA coding human AID obtained in the above Examples (SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:35) consists of introns and exons described below and comprises a full length of about 11 kb. FIG. 23 schematically shows the structure.

[0605] <SEQ ID NO:9>

[0606] Intron: Nucleotide NOs: from 1 to 1031

[0607] Exon 1: Nucleotide NOs: from 1032 to 1118

[0608] Intron: Nucleotide NOs: from 1119 to 5514

[0609] <SEQ ID NO:10>

[0610] Intron: Nucleotide NOs: from 1 to 1064

[0611] Exon 2: Nucleotide NOs: from 1065 to 1212

[0612] Intron: Nucleotide NOs: from 1213 to 2591

[0613] Exon 3: Nucleotide NOs: from 2592 to 2862

[0614] Intron: Nucleotide NOs: from 2863 to 3155

[0615] Exon 4: Nucleotide NOs: from 3156 to 3271

[0616] Intron: Nucleotide NOs: from 3272 to 3740

[0617] Exon 5: Nucleotide NOs: from 3741 to 5912

[0618] Intron: Nucleotide NOs: from 5913 to 6564

[0619] <SEQ ID NO:35>

[0620] Intron: Nucleotide NOs: from 1 to 441

[0621] Exon 1: Nucleotide NOs: from 442 to 528

[0622] Intron: Nucleotide NOs: from 529 to 6279

[0623] Exon 2: Nucleotide NOs: from 6280 to 6427

[0624] Intron: Nucleotide NOs: from 6428 to 7806

[0625] Exon 3: Nucleotide NOs: from 7807 to 8077

[0626] Intron: Nucleotide NOs: from 8078 to 8370

[0627] Exon 4: Nucleotide NOs: from 8371 to 8486

[0628] Intron: Nucleotide NOs: from 8487 to 8955

[0629] Exon 5: Nucleotide NOs: from 8956 to 11067

[0630] Intron: Nucleotide NOs: from 11068 to 11204

EXAMPLE 18 Amplification of a Given Partial Nucleotide Sequence of Genomic DNA Coding Human AID Protein by PCR and Diagnosis for the Presence or Absence of Mutation in the Partial Nucleotide Sequence

[0631] The AID protein of the present invention may involve in sideration of various immunodeficiency and allergic disease. For example, a given immunodeficiency or allergic disease may be caused by mutation or deletion in the nucleotide sequence of genomic DNA (especially exon) coding an AID protein.

[0632] The presence or absence of such mutation or deletion in genomic DNA can be analyzed by, for example, following examples.

[0633] (1) A pair of primers comprising a nucleotide sequence complementary to a given partial nucleotide sequence of genomic DNA coding AID protein in the present invention is prepared.

[0634] (2) Using genomic DNA coding AID protein obtained from tissues or cells of a patient suffering from immunodeficiency or allergic disease as a template, an objective partial nucleotide sequence of the genomic DNA is amplified with the pair of primer DNA.

[0635] (3) By analyzing the presence or absence of a PCR product and a nucleotide sequence of the PCR product, and comparing the nucleotide sequence with a corresponding nucleotide sequence in genomic DNA coding AID protein derived from a normal person, mutation or deletion in the genomic DNA is identified.

[0636] Specifically, this method enables, for example, not only elucidate relationship between immunodeficiency or allergic disease and AID protein, but also diagnose the diseases by the above method in the case that AID protein is a cause of sideration of a given type of disease (for example immunodeficiency or allergic disease).

[0637] For the above purpose, the following 15 kinds of primers were designed and prepared based on a given partial nucleotide sequence in the genomic DNA coding human AID protein.

[0638] Primer: p3 (SEQ ID No. 20)

[0639] Primer: p9 (SEQ ID No. 21)

[0640] Primer: p10 (SEQ ID No. 22)

[0641] Primer: p12 (SEQ ID No. 23)

[0642] Primer: p14 (SEQ ID No. 24)

[0643] Primer: p16 (SEQ ID No. 25)

[0644] Primer: p17 (SEQ ID No. 26)

[0645] Primer: p19 (SEQ ID No. 27)

[0646] Primer: p26 (SEQ ID No. 28)

[0647] Primer: p29 (SEQ ID No. 29)

[0648] Primer: p36 (SEQ ID No. 30)

[0649] Primer: p48 (SEQ ID No. 31)

[0650] Primer: p59 (SEQ ID No. 32)

[0651] Primer: p85 (SEQ ID No. 33)

[0652] Primer: p86 (SEQ ID No. 34)

[0653] By PCR using the above primers as a pair of primers by the following combinations, and a genomic DNA isolated from human B lymphoma cell RAMOS as a template, a partial nucleotide sequence coding each target human AID protein was amplified. FIG. 21 shows relative locations of genomic DNA partial nucleotide sequences amplified by each primer pair.

[0654] (1) DNA comprising nucleotide sequence of SEQ ID NO:31 and DNA comprising nucleotide sequence of SEQ ID NO:32;

[0655] (2) DNA comprising nucleotide sequence of SEQ ID NO:20 and DNA comprising nucleotide sequence of SEQ ID NO:22;

[0656] (3) DNA comprising nucleotide sequence of SEQ ID NO:21 and DNA comprising nucleotide sequence of SEQ ID NO:30;

[0657] (4) DNA comprising nucleotide sequence of SEQ ID NO:24 and DNA comprising nucleotide sequence of SEQ ID NO:25;

[0658] (5) DNA comprising nucleotide sequence of SEQ ID NO:23 and DNA comprising nucleotide sequence of SEQ ID NO:27;

[0659] (6) DNA comprising nucleotide sequence of SEQ ID NO:23 and DNA comprising nucleotide sequence of SEQ ID NO:28;

[0660] (7) DNA comprising nucleotide sequence of SEQ ID NO:23 and DNA comprising nucleotide sequence of SEQ ID NO:29;

[0661] (8) DNA comprising nucleotide sequence of SEQ ID NO:26 and DNA comprising nucleotide sequence of SEQ ID NO:27;

[0662] (9) DNA comprising nucleotide sequence of SEQ ID NO:26 and DNA comprising nucleotide sequence of SEQ ID NO:28;

[0663] (10) DNA comprising nucleotide sequence of SEQ ID NO:26 and DNA comprising nucleotide sequence of SEQ ID NO:29;

[0664] (11) DNA comprising nucleotide sequence of SEQ ID NO:34 and DNA comprising nucleotide sequence of SEQ ID NO:28;

[0665] (12) DNA comprising nucleotide sequence of SEQ ID NO:34 and DNA comprising nucleotide sequence of SEQ ID NO:29;

[0666] (13) DNA comprising nucleotide sequence of SEQ ID NO:33 and DNA comprising nucleotide sequence of SEQ ID NO:29; or,

[0667] (14) DNA comprising nucleotide sequence of SEQ ID NO:18 and DNA comprising nucleotide sequence of SEQ ID NO:19;

[0668] The condition for PCR was set by the following manner.

[0669] <Reaction Solution>

[0670] A total amount of 20.2 μl consisting of DDW (8 μl), 10×buffer (2 μl), dNTP (2.5 mM each, 2 μl), 2 mM primer 1 (2 μl), 2 μM primer 2 (2 μl), genomic DNA isolated from human B Lymphoma cells (185 ng/μl) and Taq polymerase (5 U/ml, 0.2 μl), Ex Taq (TAKARA), or Ampli Taq (Perkin Elmer).

[0671] <Reaction>

[0672] Reaction was conducted by the following (A) or (B).

[0673] (A) 1 cycle (reaction at 94° C. for 30 sec) and 40 cycles (reaction at 94° C. for 10 sec, reaction at 54° C. for 30 sec, and reaction at 72° C. for 3 min and 30 sec) and stored at 4° C.

[0674] (B) 1 cycle (reaction at 94° C. for 30 sec) and 40 cycles (reaction at 94° C. for 10 sec, reaction at 55° C. for 30 sec, and reaction at 72° C. for 2 min and 10 sec) and stored at 4° C.

[0675] <PCR Equipment>

[0676] A commercial PCR device (Perkin Elmer Thermal Cycler 9700 type) was used.

EXAMPLE 19 The Expression of Human AID mRNA in Various Human Organ Tissues

[0677] The expression of human AID mRNA in various human organ tissues was analyzed by RT-PCR according to the standard method (Immunity, 9:1-10, 1998).

[0678] RT-PCR was conducted by using various tissues set in the human tissue cDNA panel (CLONTECH) as a template according to the standard method.

[0679] AID cDNA was amplified by primers p17 (SEQ ID NO:26) and p26 (SEQ ID NO:28) prepared in the above and Taq polymerase.

[0680] As a control, RT-PCR in the same manner was conducted using cDNA of G3PDH as a template and GF primer and GR primer (Immunity, 9:1-10, 1998).

[0681]FIG. 24 shows the result. As a result, Specific expression of mRNA was confirmed in lymph node and tonsil. This result was consistent with the experimental result in which the expression of mRNA for mouse AID was observed in the various lymphatic tissues (Examples 8 and 9).

[0682] On the other hand, when RT-PCR was conducted with the saturated cycle number in the same manner as the above in the above RT-PCR, the expression of AID mRNA was observed in almost all analyzed organs.

EXAMPLE 20 Localization of Human AID Gene on Human Chromosomes

[0683] Localization of human AID gene on human chromosomes was analyzed by Fluorescence in situ hybridization (FISH) method (Experimental Medicine, Suppl. “Genetic Engineering Hand Book” published by Yodosha, 1992, p. 271-277).

[0684] Genomic DNA including human AID gene (exon 1 to exon 5), isolated in the above, which was labeled with biotin-11-dUTP (Sigma) by the nick translation method was used as a probe for hybridization.

[0685] The probe was hybridized with chromosomes in metaphase human cells. Hybridization signals were detected using fluorescein isothiocyanate-avidin (DCS) (Vector Laboratories).

[0686]FIG. 25 shows the result. As a result, human AID gene was found to be localized on chromosome 12p13. Moreover, this location was revealed to be near 12p13.1 which is the location for the above APOBEC-1 comprising a relatively high amino acid sequence homology with the AID protein and has the same cytidine deaminase activity as that of the AID protein.

[0687] It has been reported that some abnormality on human chromosome locus 12p13.3-12p11.2, 12p13.2-12p24.1 and 12p13 may be involved in diseases such as acrocallosal syndrome, inflammatory bowel syndrome familial periodic fever, respectively, however, causative gene thereof has not been traced yet. It has been suggested that human AID gene of the present invention may be involved in sideration of such diseases.

[0688] All references and patents cited herein are incorporated by reference in their entirety.

[0689] Industrial Applicability

[0690] The AID protein of the present invention can be considered to have a function of regulating various biological mechanisms required for generation of antigen-specific immunoglobulins (specific antibodies), which eliminate non-self antigen (foreign antigen, self-reacting cells, etc.) that triggers various diseases. More specifically, the AID protein of the present invention can be considered to be one of the enzyme that plays an important role in the genetic editing such as RNA editing and so on occurring in germinal center B cells, such as activation of B cells, class switch recombination of immunoglobulin gene, somatic hypermutation, and affinity maturation, which are the mechanisms for generation of immunoglobulin having high specificity to antigens.

[0691] The dysfunction of the AID protein of the present invention can be the cause for the humoral immunodeficiency since it induces failure of germinal center B cell function, such as antigen-specific B cell activation, class switch recombination, and somatic mutation. Reversely, the breakdown of the regulation of AID protein may induce allergy disease or autoimmune disease since it can cause inappropriate B cell activation and needless class switch recombination and somatic mutation.

[0692] Therefore, regulation of the function of AID protein and the gene encoding it enables preventing and treating various immunodeficiencies, autoimmune diseases, and allergies, which result from, for example, B cell dysfunctions (e.g. IgA deficiency, IgA nephropathy, γ globulinemia, hyper IgM syndrome, etc.) or class switch deficiency of immunoglobulin. Thus, the AID protein and the gene encoding the AID protein can be targets for the development of drugs for therapy of diseases mentioned above.

[0693] Examples of diseases whose onset prevention, symptom remission, therapy and/or symptomatic treatment effect is expected by regulating the function of the AID protein of the present invention or the gene encoding it include, for example, primary immunodeficiency syndrome with congenital disorder of immune system, mainly immunodeficiency considered to develop by B cell deficiency, decrease, or dysfunction (e.g. sex-linked agammaglobulinemia, sex-linked agammaglobulinemia with growth hormone deficiency, immunoglobulin deficiency with high IgM level, selective IgM deficiency, selective IgE deficiency, immunoglobulin heavy chain gene deletion, κ chain deficiency, IgA deficiency, IgG subclass selective deficiency, CVID (common variable immunodeficiency), infantile transient dysgammaglobulinemia, Rosen syndrome, severe combined immunodeficiency (sex-linked, autosomal recessive), ADA (adenosine deaminase) deficiency, PNP (purine nucleoside phosphorylase) deficiency, MHC class II deficiency, reticular dysplasia, Wiskott-Aldrich syndrome, ataxia telangiectasia, DiGeorge syndrome, chromosomal aberration, familial Ig hypermetabolism, hyper IgE syndrome, Gitlin syndrome, Nezelof syndrome, Good syndrome, osteodystrophy, transcobalamin syndrome, secretary bead syndrome, etc.), various diseases with antibody production deficiency that are secondary immunodeficiency syndrome with disorder of immune system caused by an acquired etiology (for example, AIDS, etc.), and/or various allergic diseases (e.g., bronchial asthma, atopic dermatitis, conjunctivitis, allergic rhinitis, allergic enteritis, drug-induced allergy, food allergy, allergic urticaria, glomerulonephritis, etc.). These could be targets for drug development.

[0694] Namely, the AID protein of the present invention, a fragment thereof, a DNA encoding the AID protein, a fragment thereof, and an antibody against the AID protein are useful as reagents for developing drugs for prevention and therapy of such diseases.

[0695] Also, the DNA itself is useful as an antisense drug regulating the function of AID gene at a gene level and in a use in gene therapy. The protein or the fragments thereof (e.g. enzyme active site) itself is useful as a drug.

[0696] Furthermore, an antibody reactive to the AID protein of the present invention or a fragment thereof is extremely useful as an antibody drug by regulating functions of the AID protein.

[0697] Furthermore, the gene (DNA), protein, and antibody of the present invention are useful as reagents for searching substrates (e.g., RNA, etc.) interacting (binding) with the protein (enzyme) of the present invention, or other auxiliary proteins associated with the protein of the present invention, and for developing drugs targeting the substrates and auxiliary proteins.

[0698] Furthermore, a method for identifying a substance that regulates production of the AID protein of the present invention or transcription of a gene encoding the AID protein into mRNA are extremely useful as means to develop drugs for therapy and prevention of various diseases (especially, immunodeficiency and allergic disease) in which the above-mentioned AID protein or AID gene is considered to be involved.

1 36 1 2440 DNA Mus musculus CDS (93)...(686) 5′UTR (1)...(92) 3′UTR (690)...(2440) misc_feature (1)...(2440) n = A,T,C or G 1 ggcacgagca gcactgaagc agccttgctt gaagcaagct tcctttggcc taagactttg 60 agggagtcaa gaaagtcacg ctggagaccg at atg gac agc ctt ctg atg aag 113 Met Asp Ser Leu Leu Met Lys 1 5 caa aag aag ttt ctt tac cat ttc aaa aat gtc cgc tgg gcc aag gga 161 Gln Lys Lys Phe Leu Tyr His Phe Lys Asn Val Arg Trp Ala Lys Gly 10 15 20 cgg cat gag acc tac ctc tgc tac gtg gtg aag agg aga gat agt gcc 209 Arg His Glu Thr Tyr Leu Cys Tyr Val Val Lys Arg Arg Asp Ser Ala 25 30 35 acc tcc tgc tca ctg gac ttc ggc cac ctt cgc aac aag tct ggc tgc 257 Thr Ser Cys Ser Leu Asp Phe Gly His Leu Arg Asn Lys Ser Gly Cys 40 45 50 55 cac gtg gaa ttg ttg ttc cta cgc tac atc tca gac tgg gac ctg gac 305 His Val Glu Leu Leu Phe Leu Arg Tyr Ile Ser Asp Trp Asp Leu Asp 60 65 70 ccg ggc cgg tgt tac cgc gtc acc tgg ttc acc tcc tgg agc ccg tgc 353 Pro Gly Arg Cys Tyr Arg Val Thr Trp Phe Thr Ser Trp Ser Pro Cys 75 80 85 tat gac tgt gcc cgg cac gtg gct gag ttt ctg aga tgg aac cct aac 401 Tyr Asp Cys Ala Arg His Val Ala Glu Phe Leu Arg Trp Asn Pro Asn 90 95 100 ctc agc ctg agg att ttc acc gcg cgc ctc tac ttc tgt gaa gac cgc 449 Leu Ser Leu Arg Ile Phe Thr Ala Arg Leu Tyr Phe Cys Glu Asp Arg 105 110 115 aag gct gag cct gag ggg ctg cgg aga ctg cac cgc gct ggg gtc cag 497 Lys Ala Glu Pro Glu Gly Leu Arg Arg Leu His Arg Ala Gly Val Gln 120 125 130 135 atc ggg atc atg acc ttc aaa gac tat ttt tac tgc tgg aat aca ttt 545 Ile Gly Ile Met Thr Phe Lys Asp Tyr Phe Tyr Cys Trp Asn Thr Phe 140 145 150 gta gaa aat cgt gaa aga act ttc aaa gcc tgg gaa ggg cta cat gaa 593 Val Glu Asn Arg Glu Arg Thr Phe Lys Ala Trp Glu Gly Leu His Glu 155 160 165 aat tct gtc cgg cta acc aga caa ctt cgg cgc atc ctt ttg ccc ttg 641 Asn Ser Val Arg Leu Thr Arg Gln Leu Arg Arg Ile Leu Leu Pro Leu 170 175 180 tac gaa gtc gat gac ttg cga gat gca ttt cgt atg ttg gga ttt 686 Tyr Glu Val Asp Asp Leu Arg Asp Ala Phe Arg Met Leu Gly Phe 185 190 195 tgaaagcaac ctcctggaat gtcacacgtg atgaaatttc tctgaagaga ctggatagaa 746 aaacaaccct tcaactacat gtttttcttc ttaagtactc acttttataa gtgtaggggg 806 aaattatatg actttttaaa aaatacttga gctgcacagg accgccagag caatgatgta 866 actgagcttg ctgtgcaaca tcgccatcta ctggggaaca gcataacttc cagactttgg 926 gtcgtgaatg atgctctttt ttttcaacag catggaaaag catatggaga cgaccacaca 986 gtttgttaca cccaccctgt gttccttgat tcatttgaat tctcaggggt atcagtgacg 1046 gattcttcta ttctttccct ctaaggctca ctttcagggg tccttttctg acaaggtcac 1106 ggggctgtcc tacagtctct gtctgagcaa tcacaagcca ttctctcaaa aacattaata 1166 ctcaggcaca tgctgtatgt tttcactgtc cgtcgtgttt ttcacatttg tatgtgaaag 1226 ggcttggggt gggatttgaa gaatgcacga tcgcctctgg gtgatttcaa taaaggatct 1286 taaaatgcag atgaggacta cgaagaaatc actctgaaaa tgagttcacg cctcaagaag 1346 caaatcccct ggaaacacag actctttttc atttttaatg tcattagttt actcacagtc 1406 ttatcaagaa gaagagttca agggttcaac ccaattttca gatcgcgtcc cttaaacatc 1466 agtaattctg ttaaagggat caaacatcct tatttcttaa ctaactggtg ccttgctgta 1526 gagaaaggag caaagcgccc agatccaaag tatatagtta tcatagccag gaaccgctac 1586 tcgttttcca ttacaaatgg caaattcttc cccgggctct cctcatagtg cctgagacgg 1646 accacggagg tgatgaacct ccggattctc tggcccaaca cggtggaagc tctgcaaggg 1706 cgcagagaca gaatgcggca gaaattgccc ccgagtccca actctccttt ccttgcgacc 1766 ttgggaacaa gacttaaagg agcctgtgac ttagaaactt ctagtaatgg gtacctggga 1826 gtcgtttgag tatggggcag tgatttattc tctgtgatgg atgccaacac ggttaaacag 1886 aatttttagt ttttatatgt gtgtgatgct gctcccccaa attgttaact gtgtaagagg 1946 gtggcaaaat agggaaagtg gcattcacct atagttccag cattcaggaa gctgaggcag 2006 gaggattgta aatttgaggc cagtctgagc tgtaaggtga gaccctattt caaacaacac 2066 agccagaatt gggttctggt aaatcatact taacaaggga aaaatgcaag acgcaagacc 2126 gtggcaagga aatgacgctt tgcccaacga aatgtaggaa accaacatag actcccagtt 2186 tgtccctctt tatgtctggt ctccctaaca acgatctttg ctaatgagaa aaatattaga 2246 aaaaaatatc cctgtgcaat tatcacccag tcgccattat aatgcaatta aaaggcccac 2306 aagaaatcct gtatacacga ccgttattta ttgtatgtaa gttgctgagg aagaggagaa 2366 aaaaataaag atcatccatt ccttcctgca aaaaaaaaaa aaaaaanaaa aaaaaaaaaa 2426 aaaaaaaaaa aaaa 2440 2 198 PRT Mus musculus 2 Met Asp Ser Leu Leu Met Lys Gln Lys Lys Phe Leu Tyr His Phe Lys 1 5 10 15 Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val 20 25 30 Val Lys Arg Arg Asp Ser Ala Thr Ser Cys Ser Leu Asp Phe Gly His 35 40 45 Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr 50 55 60 Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp 65 70 75 80 Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Glu 85 90 95 Phe Leu Arg Trp Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg 100 105 110 Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg 115 120 125 Leu His Arg Ala Gly Val Gln Ile Gly Ile Met Thr Phe Lys Asp Tyr 130 135 140 Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn Arg Glu Arg Thr Phe Lys 145 150 155 160 Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Thr Arg Gln Leu 165 170 175 Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala 180 185 190 Phe Arg Met Leu Gly Phe 195 3 30 DNA Artificial Sequence Artificially synthesized primer sequence, AID138 3 ggaattcgcc atggacagcc ttctgatgaa 30 4 30 DNA Artificial Sequence Artificially synthesized primer sequence, AID161 4 gccgctcgag tcaaaatccc aacatacgaa 30 5 25 DNA Artificial Sequence Artificially synthesized primer sequence, AID118 5 ggctgaggtt agggttccat ctcag 25 6 25 DNA Artificial Sequence Artificially synthesized primer sequence, AID119 6 gagggagtca agaaagtcac gctgg 25 7 2818 DNA Homo sapiens CDS (80)...(673) 5′UTR (1)...(79) 3′UTR (677)...(2818) 7 agagaaccat cattaattga agtgagattt ttctggcctg agacttgcag ggaggcaaga 60 agacactctg gacaccact atg gac agc ctc ttg atg aac cgg agg aag ttt 112 Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe 1 5 10 ctt tac caa ttc aaa aat gtc cgc tgg gct aag ggt cgg cgt gag acc 160 Leu Tyr Gln Phe Lys Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr 15 20 25 tac ctg tgc tac gta gtg aag agg cgt gac agt gct aca tcc ttt tca 208 Tyr Leu Cys Tyr Val Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser 30 35 40 ctg gac ttt ggt tat ctt cgc aat aag aac ggc tgc cac gtg gaa ttg 256 Leu Asp Phe Gly Tyr Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu 45 50 55 ctc ttc ctc cgc tac atc tcg gac tgg gac cta gac cct ggc cgc tgc 304 Leu Phe Leu Arg Tyr Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys 60 65 70 75 tac cgc gtc acc tgg ttc acc tcc tgg agc ccc tgc tac gac tgt gcc 352 Tyr Arg Val Thr Trp Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala 80 85 90 cga cat gtg gcc gac ttt ctg cga ggg aac ccc aac ctc agt ctg agg 400 Arg His Val Ala Asp Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg 95 100 105 atc ttc acc gcg cgc ctc tac ttc tgt gag gac cgc aag gct gag ccc 448 Ile Phe Thr Ala Arg Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro 110 115 120 gag ggg ctg cgg cgg ctg cac cgc gcc ggg gtg caa ata gcc atc atg 496 Glu Gly Leu Arg Arg Leu His Arg Ala Gly Val Gln Ile Ala Ile Met 125 130 135 acc ttc aaa gat tat ttt tac tgc tgg aat act ttt gta gaa aac cat 544 Thr Phe Lys Asp Tyr Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His 140 145 150 155 gaa aga act ttc aaa gcc tgg gaa ggg ctg cat gaa aat tca gtt cgt 592 Glu Arg Thr Phe Lys Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg 160 165 170 ctc tcc aga cag ctt cgg cgc atc ctt ttg ccc ctg tat gag gtt gat 640 Leu Ser Arg Gln Leu Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp 175 180 185 gac tta cga gac gca ttt cgt act ttg gga ctt tgatagcaac ttccaggaat 693 Asp Leu Arg Asp Ala Phe Arg Thr Leu Gly Leu 190 195 gtcacacacg atgaaatatc tctgctgaag acagtggata aaaaacagtc cttcaagtct 753 tctctgtttt tattcttcaa ctctcacttt cttagagttt acagaaaaaa tatttatata 813 cgactcttta aaaagatcta tgtcttgaaa atagagaagg aacacaggtc tggccaggga 873 cgtgctgcaa ttggtgcagt tttgaatgca acattgtccc ctactgggaa taacagaact 933 gcaggacctg ggagcatcct aaagtgtcaa cgtttttcta tgacttttag gtaggatgag 993 agcagaaggt agatcctaaa aagcatggtg agaggatcaa atgtttttat atcaacatcc 1053 tttattattt gattcatttg agttaacagt ggtgttagtg atagattttt ctattctttt 1113 cccttgacgt ttactttcaa gtaacacaaa ctcttccatc aggccatgat ctataggacc 1173 tcctaatgag agtatctggg tgattgtgac cccaaaccat ctctccaaag cattaatatc 1233 caatcatgcg ctgtatgttt taatcagcag aagcatgttt ttatgtttgt acaaaagaag 1293 attgttatgg gtggggatgg aggtatagac catgcatggt caccttcaag ctactttaat 1353 aaaggatctt aaaatgggca ggaggactgt gaacaagaca ccctaataat gggttgatgt 1413 ctgaagtagc aaatcttctg gaaacgcaaa ctcttttaag gaagtcccta atttagaaac 1473 acccacaaac ttcacatatc ataattagca aacaattgga aggaagttgc ttgaatgttg 1533 gggagaggaa aatctattgg ctctcgtggg tctcttcatc tcagaaatgc caatcaggtc 1593 aaggtttgct acattttgta tgtgtgtgat gcttctccca aaggtatatt aactatataa 1653 gagagttgtg acaaaacaga atgataaagc tgcgaaccgt ggcacacgct catagttcta 1713 gctgcttggg aggttgagga gggaggatgg cttgaacaca ggtgttcaag gccagcctgg 1773 gcaacataac aagatcctgt ctctcaaaaa aaaaaaaaaa aaaaagaaag agagagggcc 1833 gggcgtggtg gctcacgcct gtaatcccag cactttggga ggccgagccg ggcggatcac 1893 ctgtggtcag gagtttgaga ccagcctggc caacatggca aaaccccgtc tgtactcaaa 1953 atgcaaaaat tagccaggcg tggtagcagg cacctgtaat cccagctact tgggaggctg 2013 aggcaggaga atcgcttgaa cccaggaggt ggaggttgca gtaagctgag atcgtgccgt 2073 tgcactccag cctgggcgac aagagcaaga ctctgtctca gaaaaaaaaa aaaaaaagag 2133 agagagagag aaagagaaca atatttggga gagaaggatg gggaagcatt gcaaggaaat 2193 tgtgctttat ccaacaaaat gtaaggagcc aataagggat ccctatttgt ctcttttggt 2253 gtctatttgt ccctaacaac tgtctttgac agtgagaaaa atattcagaa taaccatatc 2313 cctgtgccgt tattacctag caacccttgc aatgaagatg agcagatcca caggaaaact 2373 tgaatgcaca actgtcttat tttaatctta ttgtacataa gtttgtaaaa gagttaaaaa 2433 ttgttacttc atgtattcat ttatatttta tattattttg cgtctaatga ttttttatta 2493 acatgatttc cttttctgat atattgaaat ggagtctcaa agcttcataa atttataact 2553 ttagaaatga ttctaataac aacgtatgta attgtaacat tgcagtaatg gtgctacgaa 2613 gccatttctc ttgattttta gtaaactttt atgacagcaa atttgcttct ggctcacttt 2673 caatcagtta aataaatgat aaataatttt ggaagctgtg aagataaaat accaaataaa 2733 ataatataaa agtgatttat atgaagttaa aataaaaaat cagtatgatg gaataaactt 2793 gaaaaaaaaa aaaaaaaaaa aaaaa 2818 8 198 PRT Homo sapiens 8 Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys 1 5 10 15 Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val 20 25 30 Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr 35 40 45 Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr 50 55 60 Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp 65 70 75 80 Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp 85 90 95 Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg 100 105 110 Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg 115 120 125 Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr 130 135 140 Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys 145 150 155 160 Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu 165 170 175 Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala 180 185 190 Phe Arg Thr Leu Gly Leu 195 9 5514 DNA Homo sapiens intron (1)...(1031) exon (1032)...(1118) intron (1119)...(5514) 9 acagacgaat acatggtcca agctagggct attgatttga aaatcatcaa ggtatagatg 60 gtatcaaagg cttgaggcag gaagagagca gagaccctag ctgcattgct tagcattgca 120 tccctagcac ctggcatagt ttccattaac agtaggcatg aagtatctac tcagtgaata 180 aatagaatgc atatgggcta cagtaggaga gagaaataaa atctttaata gaccaagttc 240 tatgagagca caaaattaaa gtcttttatt tgaagatctt agcctgtttt ccaaattcag 300 tgcagccagt tagacactga ttctgtctgg tgaaacaagc atttttgtat tttgggggac 360 tgctgctgct tctgactcca aattaaggat tttttttttt tctaaaaaag atggctcatg 420 caaaaatcac tctttggtgt aaatatctag tcttcaagca attcttgtaa tgcaatcaga 480 aagaaaaaaa tccatggttt gggaggcaaa atttttgtgt tctaaattct atataactga 540 gttcatttgc ttaactgcaa agcaggagct gctagtgcct gtctgtactg aggttcagag 600 agactgtggg aatatggggg aattagaggc tatctgaggc tcttcaacac aataacccaa 660 gaagctattt aaatgctctt taaggtattt acataaatat tactattctc attgtgcttt 720 tattttgtgt tatcatgatt ataattgaag tgtctactgt tactgcctcc tgatctttgc 780 tagctatgga gcatggactg ggcttttaga gcagcagccc caaaggaacc taaacattaa 840 agcagagctg ccctcaatgg tttaacctgt gtgactctgc ctatgacagc cccacccacc 900 catcttcact ggatccaaat caggagcaag gccgttgggg tacctggtgg gggtgatgct 960 gtcaggggag gagcccaaaa gggcaagctc aaatttgaat gtgaagggcc aatgcactgt 1020 cagactgaga cagagaacca tcattaattg aagtgagatt tttctggcct gagacttgca 1080 gggaggcaag aagacactct ggacaccact atggacaggt aaagaggcag tcttctcgtg 1140 ggtgattgca ctggccttcc tctcagagca aatctgagta atgagactgg tagctatccc 1200 tttctctcat gtaactgtct gactgataag atcagcttga tcaatatgca tatatatttt 1260 ttgatctgtc tccttttctt ctattcagat cttatacgct gtcagcccaa ttctttctgt 1320 ttcagacttc tcttgatttc cctctttttc atgtggcaaa agaagtagtg cgtacaatgt 1380 actgattcgt cctgagattt gtaccatggt tgaaactaat ttatggtaat aatattaaca 1440 tagcaaatct ttagagactc aaatcatgaa aaggtaatag cagtactgta ctaaaaacgg 1500 tagtgctaat tttcgtaata attttgtaaa tattcaacag taaaacaact tgaagacaca 1560 ctttcctagg gaggcgttac tgaaataatt tagctatagt aagaaaattt gtaattttag 1620 aaatgccaag cattctaaat taattgcttg aaagtcacta tgattgtgtc cattataagg 1680 agacaaattc attcaagcaa gttatttaat gttaaaggcc caattgttag gcagttaatg 1740 gcacttttac tattaactaa tctttccatt tgttcagacg tagcttaact tacctcttag 1800 gtgtgaattt ggttaaggtc ctcataatgt ctttatgtgc agtttttgat aggttattgt 1860 catagaactt attctattcc tacatttatg attactatgg atgtatgaga ataacaccta 1920 atccttatac tttacctcaa tttaactcct ttataaagaa cttacattac agaataaaga 1980 ttttttaaaa atatattttt ttgtagagac agggtcttag cccagccgag gctggtctct 2040 aagtcctggc ccaagcgatc ctcctgcctg ggcctcctaa agtgctggaa ttatagacat 2100 gagccatcac atccaatata cagaataaag atttttaatg gaggatttaa tgttcttcag 2160 aaaattttct tgaggtcaga caatgtcaaa tgtctcctca gtttacactg agattttgaa 2220 aacaagtctg agctataggt ccttgtgaag ggtccattgg aaatacttgt tcaaagtaaa 2280 atggaaagca aaggtaaaat cagcagttga aattcagaga aagacagaaa aggagaaaag 2340 atgaaattca acaggacaga agggaaatat attatcatta aggaggacag tatctgtaga 2400 gctcattagt gatggcaaaa tgacttggtc aggattattt ttaacccgct tgtttctggt 2460 ttgcacggct ggggatgcag ctagggttct gcctcaggga gcacagctgt ccagagcagc 2520 tgtcagcctg caagcctgaa acactccctc ggtaaagtcc ttcctactca ggacagaaat 2580 gacgagaaca gggagctgga aacaggcccc taaccagaga agggaagtaa tggatcaaca 2640 aagttaacta gcaggtcagg atcacgcaat tcatttcact ctgactggta acatgtgaca 2700 gaaacagtgt aggcttattg tattttcatg tagagtagga cccaaaaatc cacccaaagt 2760 cctttatcta tgccacatcc ttcttatcta tacttccagg acactttttc ttccttatga 2820 taaggctctc tctctctcca cacacacaca cacacacaca cacacacaca cacacacaca 2880 cacaaacaca caccccgcca accaaggtgc atgtaaaaag atgtagattc ctctgccttt 2940 ctcatctaca cagcccagga gggtaagtta atataagagg gatttattgg taagagatga 3000 tgcttaatct gtttaacact gggcctcaaa gagagaattt cttttcttct gtacttatta 3060 agcacctatt atgtgttgag cttatatata caaagggtta ttatatgcta atatagtaat 3120 agtaatgktg gttggtacta tggtaattac cataaaaatt awtatccttt taaaataaag 3180 ctaattatta ttggatcttt tttagtattc attttatgtt ttttatgttt ttgatttttt 3240 aaaagacaat ctcaccctgt tacccaggct ggagtgcagt ggtgcaatca tagctttctg 3300 cagtcttgaa ctcctgggct caagcaatcc tcctgccttg gcctcccaaa gtgttgggat 3360 acagtcatga gccactgcat ctggcctagg atccatttag attaaaatat gcattttaaa 3420 ttttaaaata atatggctaa tttttacctt atgtaatgtg tatactggta ataaatctag 3480 tttgctgcct aaagtttaaa gtgctttcca ataagcttca tgtacgtgag gggagacatt 3540 taaagtgaaa cagacagcca ggtgtggtgg ctcacgcctg taatcccagc actctgggag 3600 gctgaggtgg gtggatcgct tgagccctgg agttcaagac cagcctgagc aacatggcaa 3660 aaccctgttt ctataacaaa aattagccgg gcatggtggc atgtgcctgt ggtcccagct 3720 actagggggc tgaggcagga gaatctttgg agcccaggag gtcaaggctg cactgagcag 3780 tgcttgcgcc actgcactcc agcctgggtg acaggaccag accttgcctc aaaaaaataa 3840 gaagaaaaat taaaaataaa tggaaacaac tacaaagagc tgttgtccta gatgagctac 3900 ttagttaggc tgatattttg gtatttaact tttaaagtca gggtctgtca cctgcactac 3960 attattaaaa tatcaattct caatgtatat ccacacaaag actggtacgt gaatgttcat 4020 agtaccttta ttcacaaaac cccaaagtag agactatcca aatatccatc aacaagtgaa 4080 caaataaaca aaatgtgcta tatccatgca atggaatacc accctgcagt acaaaggaag 4140 aagctacttg gggatgaatc ccaaagtcat gacgctaaat gaaagagtca gacatgaagg 4200 aggagataat gtatgccata cgaaattcta gaaaatgaaa gtaacttata gttacagaaa 4260 gcaaatcagg gcaggcatag aggctcacac ctgtaatccc agcactttga gaggccacgt 4320 gggaagattg ctagaactca ggagttcaag accagcctgg gcaacacagt gaaactccat 4380 tctccacaaa aatgggaaaa aaagaaagca aatcagtggt tgtcctgtgg ggaggggaag 4440 gactgcaaag agggaagaag ctctggtggg gtgagggtgg tgattcaggt tctgtatcct 4500 gactgtggta gcagtttggg gtgtttacat ccaaaaatat tcgtagaatt atgcatctta 4560 aatgggtgga gtttactgta tgtaaattat acctcaatgt aagaaaaaat aatgtgtaag 4620 aaaagtttca attctcttgc cagcaaacgt tattcaaatt cctgagccct ttacttcgca 4680 aattctctgc acttctgccc cgtaccatta ggtgacagca ctagctccac aaattggata 4740 aatgcatttc tggaaaagac tagggacaaa atccaggcat cacttgtgct ttcatatcaa 4800 ccacgctgta cagcttgtgt tgctgtctgc agctgcaatg gggactcttg atttctttaa 4860 ggaaacttgg gttaccagag tatttccaca aatgctattc aaattagtgc ttatgatatg 4920 caagacactg tgctaggagc cagaaaacaa agaggaggag aaatcagtca ttatgtggga 4980 acaacatagc aagatattta gatcattttg actagttaaa aaagcagcag agtacaaaat 5040 cacacatgca atcagtataa tccaaatcat gtaaatatgt gcctgtagaa agactagagg 5100 aataaacaca agaatcttaa cagtcattgt cattagacac taagtctaat tattattatt 5160 agacactatg atatttgaga tttaaaaaat ctttaatatt ttaaaattta gagctcttct 5220 atttttccat agtattcaag tttgacaatg atcaagtatt actctttctt tttttttttt 5280 tttttttttt tttgagatgg agttttggtc ttgttgccca tgctggagtg gaatggcatg 5340 aycatagctc actgcaacct ccacctcctg ggttcaagca aagctgtcgc ctcagcctcc 5400 cgggtagatg ggattacagg cgcccaccac cacactcggc taatgtttgt atttttagta 5460 gagatggggt ttcaccatgt tggccaggct ggtctcaaac tcctgacctc agag 5514 10 6564 DNA Homo sapiens 10 gggggcctgt aatcccagct actcaggagg ctgaggcagg aggatccgcg gagcctggca 60 gatctgcctg agcctgggag gttgaggcta cagtaagcca agatcatgcc agtatacttc 120 agcctgggcg acaaagtgag accgtaacaa aaaaaaaaaa atttaaaaaa agaaatttag 180 atcaagatcc aactgtaaaa agtggcctaa acaccacatt aaagagtttg gagtttattc 240 tgcaggcaga agagaaccat cagggggtct tcagcatggg aatggcatgg tgcacctggt 300 ttttgtgaga tcatggtggt gacagtgtgg ggaatgttat tttggaggga ctggaggcag 360 acagaccggt taaaaggcca gcacaacaga taaggaggaa gaagatgagg gcttggaccg 420 aagcagagaa gagcaaacag ggaaggtaca aattcaagaa atattggggg gtttgaatca 480 acacatttag atgattaatt aaatatgagg actgaggaat aagaaatgag tcaaggatgg 540 ttccaggctg ctaggctgct tacctgaggt ggcaaagtcg ggaggagtgg cagtttagga 600 cagggggcag ttgaggaata ttgttttgat cattttgagt ttgaggtaca agttggacac 660 ttaggtaaag actggagggg aaatctgaat atacaattat gggactgagg aacaagttta 720 ttttattttt tgtttcgttt tcttgttgaa gaacaaattt aattgtaatc ccaagtcatc 780 agcatctaga agacagtggc aggaggtgac tgtcttgtgg gtaagggttt ggggtccttg 840 atgagtatct ctcaattggc cttaaatata agcaggaaaa ggagtttatg atggattcca 900 ggctcagcag ggctcaggag ggctcaggca gccagcagag gaagtcagag catcttcttt 960 ggtttagccc aagtaatgac ttccttaaaa agctgaagga aaatccagag tgaccagatt 1020 ataaactgta ctcttgcatt ttctctccct cctctcaccc acagcctctt gatgaaccgg 1080 aggaagtttc tttaccaatt caaaaatgtc cgctgggcta agggtcggcg tgagacctac 1140 ctgtgctacg tagtgaagag gcgtgacagt gctacatcct tttcactgga ctttggttat 1200 cttcgcaata aggtatcaat taaagtcagc tttgcaagca gtttaatggt caactgtgag 1260 tgcttttaga gccacctgct gatggtatta cttccatcct tttttggcat ttgtgtctct 1320 atcacattcc tcaaatcctt ttttttattt ctttttccat gtccatgcac ccatattaga 1380 catggcccaa aatatgtgat ttaattcctc cccagtaatg ctgggcaccc taataccact 1440 ccttccttca gtgccaagaa caactgctcc caaactgttt accagctttc ctcagcatct 1500 gaattgcctt tgagattaat taagctaaaa gcatttttat atgggagaat attatcagct 1560 tgtccaagca aaaattttaa atgtgaaaaa caaattgtgt cttaagcatt tttgaaaatt 1620 aaggaagaag aatttgggaa aaaattaacg gtggttcaat tctgttttcc aaatgatttc 1680 ttttccctcc tactcacatg ggtcgtaggc cagtgaatac attcaacatg gtgatcccca 1740 gaaaactcag agaagcctcg gctgatgatt aattaaattg atctttcggc tacccgagag 1800 aattacattt ccaagagact tcttcaccaa aatccagatg ggtttacata aacttctgcc 1860 catgggtatc tcctctctcc taacacgctg tgacgtctgg gcttggtgga atctcaggga 1920 agcatccgtg gggtggaagg tcatcgtctg gctcgttgtt tgatggttat attaccatgc 1980 aattttcttt gcctacattt gtattgaata catcccaatc tccttcctat tcggtgacat 2040 gacacattct atttcagaag gctttgattt tatcaagcac tttcatttac ttctcatggc 2100 agtgcctatt acttctctta caatacccat ctgtctgctt taccaaaatc tatttcccct 2160 tttcagatcc tcccaaatgg tcctcataaa ctgtcctgcc tccacctagt ggtccaggta 2220 tatttccaca atgttacatc aacaggcact tctagccatt ttccttctca aaaggtgcaa 2280 aaagcaactt cataaacaca aattaaatct tcggtgaggt agtgtgatgc tgcttcctcc 2340 caactcagcg cacttcgtct tcctcattcc acaaaaaccc atagccttcc ttcactctgc 2400 aggactagtg ctgccaaggg ttcagctcta cctactggtg tgctcttttg agcaagttgc 2460 ttagcctctc tgtaacacaa ggacaatagc tgcaagcatc cccaaagatc attgcaggag 2520 acaatgacta aggctaccag agccgcaata aaagtcagtg aattttagcg tggtcctctc 2580 tgtctctcca gaacggctgc cacgtggaat tgctcttcct ccgctacatc tcggactggg 2640 acctagaccc tggccgctgc taccgcgtca cctggttcac ctcctggagc ccctgctacg 2700 actgtgcccg acatgtggcc gactttctgc gagggaaccc caacctcagt ctgaggatct 2760 tcaccgcgcg cctctacttc tgtgaggacc gcaaggctga gcccgagggg ctgcggcggc 2820 tgcaccgcgc cggggtgcaa atagccatca tgaccttcaa aggtgcgaaa gggccttccg 2880 cgcaggcgca gtgcagcagc ccgcattcgg gattgcgatg cggaatgaat gagttagtgg 2940 ggaagctcga ggggaagaag tgggcgggga ttctggttca cctctggagc cgaaattaaa 3000 gattagaagc agagaaaaga gtgaatggct cagagacaag gccccgagga aatgagaaaa 3060 tggggccagg gttgcttctt tcccctcgat ttggaacctg aactgtcttc tacccccata 3120 tccccgcctt tttttccttt tttttttttt tgaagattat ttttactgct ggaatacttt 3180 tgtagaaaac cacgaaagaa ctttcaaagc ctgggaaggg ctgcatgaaa attcagttcg 3240 tctctccaga cagcttcggc gcatcctttt ggtaaggggc ttcctcgctt tttaaatttt 3300 ctttctttct ctacagtctt ttttggagtt tcgtatattt cttatatttt cttattgttc 3360 aatcactctc agttttcatc tgatgaaaac tttatttctc ctccacatca gctttttctt 3420 ctgctgtttc accattcaga gccctctgct aaggttcctt ttccctccct tttctttctt 3480 ttgttgtttc acatctttaa atttctgtct ctccccaggg ttgcgtttcc ttcctggtca 3540 gaattctttt ctcctttttt tttttttttt tttttttttt taaacaaaca aacaaaaaac 3600 ccaaaaaaac tctttcccaa tttactttct tccaacatgt tacaaagcca tccactcagt 3660 ttagaagact ctccggcccc accgaccccc aacctcgttt tgaagccatt cactcaattt 3720 gcttctctct ttctctacag cccctgtatg aggttgatga cttacgagac gcatttcgta 3780 ctttgggact ttgatagcaa cttccaggaa tgtcacacac gatgaaatat ctctgctgaa 3840 gacagtggat aaaaaacagt ccttcaagtc ttctctgttt ttattcttca actctcactt 3900 tcttagagtt tacagaaaaa atatttatat acgactcttt aaaaagatct atgtcttgaa 3960 aatagagaag gaacacaggt ctggccaggg acgtgctgca attggtgcag ttttgaatgc 4020 aacattgtcc cctactggga ataacagaac tgcaggacct gggagcatcc taaagtgtca 4080 acgtttttct atgactttta ggtaggatga gagcagaagg tagatcctaa aaagcatggt 4140 gagaggatca aatgttttta tatcaacatc ctttattatt tgattcattt gagttaacag 4200 tggtgttagt gatagatttt tctattcttt tcccttgacg tttactttca agtaacacaa 4260 actcttccat caggccatga tctataggac ctcctaatga gagtatctgg gtgattgtga 4320 ccccaaacca tctctccaaa gcattaatat ccaatcatgc gctgtatgtt ttaatcagca 4380 gaagcatgtt tttatgtttg tacaaaagaa gattgttatg ggtggggatg gaggtataga 4440 ccatgcatgg tcaccttcaa gctactttaa taaaggatct taaaatgggc aggaggactg 4500 tgaacaagac accctaataa tgggttgatg tctgaagtag caaatcttct ggaaacgcaa 4560 actcttttaa ggaagtccct aatttagaaa cacccacaaa cttcacatat cataattagc 4620 aaacaattgg aaggaagttg cttgaatgtt ggggagagga aaatctattg gctctcgtgg 4680 gtctcttcat ctcagaaatg ccaatcaggt caaggtttgc tacattttgt atgtgtgtga 4740 tgcttctccc aaaggtatat taactatata agagagttgt gacaaaacag aatgataaag 4800 ctgcgaaccg tggcacacgc tcatagttct agctgcttgg gaggttgagg agggaggatg 4860 gcttgaacac aggtgttcaa ggccagcctg ggcaacataa caagatcctg tctctcaaaa 4920 aaaaaaaaaa aaaaaagaaa gagagagggc cgggcgtggt ggctcacgcc tgtaatccca 4980 gcactttggg aggccgagcc gggcggatca cctgtggtca ggagtttgag accagcctgg 5040 ccaacatggc aaaaccccgt ctgtactcaa aatgcaaaaa ttagccaggc gtggtagcag 5100 gcacctgtaa tcccagctac ttgggaggct gaggcaggag aatcgcttga acccaggagg 5160 tggaggttgc agtaagctga gatcgtgccg ttgcactcca gcctgggcga caagagcaag 5220 actctgtctc agaaaaaaaa aaaaaaaaga gagagagaga gaaagagaac aatatttggg 5280 agagaaggat ggggaagcat tgcaaggaaa ttgtgcttta tccaacaaaa tgtaaggagc 5340 caataaggga tccctatttg tctcttttgg tgtctatttg tccctaacaa ctgtctttga 5400 cagtgagaaa aatattcaga ataaccatat ccctgtgccg ttattaccta gcaacccttg 5460 caatgaagat gagcagatcc acaggaaaac ttgaatgcac aactgtctta ttttaatctt 5520 attgtacata agtttgtaaa agagttaaaa attgttactt catgtattca tttatatttt 5580 atattatttt gcgtctaatg attttttatt aacatgattt ccttttctga tatattgaaa 5640 tggagtctca aagcttcata aatttataac tttagaaatg attctaataa caacgtatgt 5700 aattgtaaca ttgcagtaat ggtgctacga agccatttct cttgattttt agtaaacttt 5760 tatgacagca aatttgcttc tggctcactt tcaatcagtt aaataaatga taaataattt 5820 tggaagctgt gaagataaaa taccaaataa aataatataa aagtgattta tatgaagtta 5880 aaataaaaaa tcagtatgat ggaataaact tgagagtcca gaagttatcc catacatctg 5940 taatcaacta atttctcaca agggtgtaag gaccattcaa tggagaaaaa atgatcttct 6000 caacaaatgg tgctgagcta attggatatt acatgcaaag gaatgaattt gagtctctac 6060 tacacaccat atataaaaat taattaaaaa ttcatcaaat acctaaatat tagagactaa 6120 tttataaacc gtagagagaa acataggtaa aaatgtttat ggctttagat taggcaacag 6180 cttcttaatt atgacatcaa aagcacaagc aaccaaagac aaaaataaat cagttggact 6240 tcatcgaaat taaaaatctt tgtgcatcaa aggacactta gtaagaaagt gaaaagacaa 6300 cccacagaag tgggagaaaa cacttgcaaa tcatatatct gataagggtt gtgatattat 6360 gatatatata taggtttttg tccatagttc ctggcttata aaccccctca cccttgttac 6420 agtcatttgt tataaggttg gatggtttag gcctcagaag caaaactctc tctctcacct 6480 tctccagccc tcctgtctct ggcacctcat tcttccctga ggccacatag aaactagaat 6540 ctctcttcca caaggcggtc aaag 6564 11 87 DNA Homo sapiens 11 agagaaccat cattaattga agtgagattt ttctggcctg agacttgcag ggaggcaaga 60 agacactctg gacaccacta tggacag 87 12 148 DNA Homo sapiens 12 cctcttgatg aaccggagga agtttcttta ccaattcaaa aatgtccgct gggctaaggg 60 tcggcgtgag acctacctgt gctacgtagt gaagaggcgt gacagtgcta catccttttc 120 actggacttt ggttatcttc gcaataag 148 13 271 DNA Homo sapiens 13 aacggctgcc acgtggaatt gctcttcctc cgctacatct cggactggga cctagaccct 60 ggccgctgct accgcgtcac ctggttcacc tcctggagcc cctgctacga ctgtgcccga 120 catgtggccg actttctgcg agggaacccc aacctcagtc tgaggatctt caccgcgcgc 180 ctctacttct gtgaggaccg caaggctgag cccgaggggc tgcggcggct gcaccgcgcc 240 ggggtgcaaa tagccatcat gaccttcaaa g 271 14 116 DNA Homo sapiens 14 attattttta ctgctggaat acttttgtag aaaaccacga aagaactttc aaagcctggg 60 aagggctgca tgaaaattca gttcgtctct ccagacagct tcggcgcatc cttttg 116 15 2172 DNA Homo sapiens 15 cccctgtatg aggttgatga cttacgagac gcatttcgta ctttgggact ttgatagcaa 60 cttccaggaa tgtcacacac gatgaaatat ctctgctgaa gacagtggat aaaaaacagt 120 ccttcaagtc ttctctgttt ttattcttca actctcactt tcttagagtt tacagaaaaa 180 atatttatat acgactcttt aaaaagatct atgtcttgaa aatagagaag gaacacaggt 240 ctggccaggg acgtgctgca attggtgcag ttttgaatgc aacattgtcc cctactggga 300 ataacagaac tgcaggacct gggagcatcc taaagtgtca acgtttttct atgactttta 360 ggtaggatga gagcagaagg tagatcctaa aaagcatggt gagaggatca aatgttttta 420 tatcaacatc ctttattatt tgattcattt gagttaacag tggtgttagt gatagatttt 480 tctattcttt tcccttgacg tttactttca agtaacacaa actcttccat caggccatga 540 tctataggac ctcctaatga gagtatctgg gtgattgtga ccccaaacca tctctccaaa 600 gcattaatat ccaatcatgc gctgtatgtt ttaatcagca gaagcatgtt tttatgtttg 660 tacaaaagaa gattgttatg ggtggggatg gaggtataga ccatgcatgg tcaccttcaa 720 gctactttaa taaaggatct taaaatgggc aggaggactg tgaacaagac accctaataa 780 tgggttgatg tctgaagtag caaatcttct ggaaacgcaa actcttttaa ggaagtccct 840 aatttagaaa cacccacaaa cttcacatat cataattagc aaacaattgg aaggaagttg 900 cttgaatgtt ggggagagga aaatctattg gctctcgtgg gtctcttcat ctcagaaatg 960 ccaatcaggt caaggtttgc tacattttgt atgtgtgtga tgcttctccc aaaggtatat 1020 taactatata agagagttgt gacaaaacag aatgataaag ctgcgaaccg tggcacacgc 1080 tcatagttct agctgcttgg gaggttgagg agggaggatg gcttgaacac aggtgttcaa 1140 ggccagcctg ggcaacataa caagatcctg tctctcaaaa aaaaaaaaaa aaaaaagaaa 1200 gagagagggc cgggcgtggt ggctcacgcc tgtaatccca gcactttggg aggccgagcc 1260 gggcggatca cctgtggtca ggagtttgag accagcctgg ccaacatggc aaaaccccgt 1320 ctgtactcaa aatgcaaaaa ttagccaggc gtggtagcag gcacctgtaa tcccagctac 1380 ttgggaggct gaggcaggag aatcgcttga acccaggagg tggaggttgc agtaagctga 1440 gatcgtgccg ttgcactcca gcctgggcga caagagcaag actctgtctc agaaaaaaaa 1500 aaaaaaaaga gagagagaga gaaagagaac aatatttggg agagaaggat ggggaagcat 1560 tgcaaggaaa ttgtgcttta tccaacaaaa tgtaaggagc caataaggga tccctatttg 1620 tctcttttgg tgtctatttg tccctaacaa ctgtctttga cagtgagaaa aatattcaga 1680 ataaccatat ccctgtgccg ttattaccta gcaacccttg caatgaagat gagcagatcc 1740 acaggaaaac ttgaatgcac aactgtctta ttttaatctt attgtacata agtttgtaaa 1800 agagttaaaa attgttactt catgtattca tttatatttt atattatttt gcgtctaatg 1860 attttttatt aacatgattt ccttttctga tatattgaaa tggagtctca aagcttcata 1920 aatttataac tttagaaatg attctaataa caacgtatgt aattgtaaca ttgcagtaat 1980 ggtgctacga agccatttct cttgattttt agtaaacttt tatgacagca aatttgcttc 2040 tggctcactt tcaatcagtt aaataaatga taaataattt tggaagctgt gaagataaaa 2100 taccaaataa aataatataa aagtgattta tatgaagtta aaataaaaaa tcagtatgat 2160 ggaataaact tg 2172 16 25 DNA Artificial Sequence Artificially synthesized primer sequence, 170 16 gagaccgata tggacagcct tctga 25 17 27 DNA Artificial Sequence Artificially synthesized primer sequence, 181 17 tcacgtgtga cattccagga ggttgct 27 18 30 DNA Artificial Sequence Artificially synthesized primer sequence, 22 18 gtagtgaaga ggcgtgacag tgctacatcc 30 19 27 DNA Artificial Sequence Artificially synthesized primer sequence, 25 19 gttccctcgc agaaagtcgg ccacatg 27 20 24 DNA Artificial Sequence Artificially synthesized primer sequence, p3 20 gagtttgagg tacaagttgg acac 24 21 23 DNA Artificial Sequence Artificially synthesized primer sequence, p9 21 tatctcctct ctcctaacac gct 23 22 23 DNA Artificial Sequence Artificially synthesized primer sequence, p10 22 acaagctgat aatattctcc cat 23 23 22 DNA Artificial Sequence Artificially synthesized primer sequence, p12 23 tcttcggtga ggtagtgtga tg 22 24 30 DNA Artificial Sequence Artificially synthesized primer sequence, p14 24 agcctcttga tgaaccggag gaagtttctt 30 25 28 DNA Artificial Sequence Artificially synthesized primer sequence, p16 25 ttattgcgaa gataaccaaa gtccagtg 28 26 21 DNA Artificial Sequence Artificially synthesized primer sequence, p17 26 tagaccctgg ccgctgctac c 21 27 22 DNA Artificial Sequence Artificially synthesized primer sequence, p19 27 cgcatcgcaa tcccgaatgc gg 22 28 28 DNA Artificial Sequence Artificially synthesized primer sequence, p26 28 caaaaggatg cgccgaagct gtctggag 28 29 23 DNA Artificial Sequence Artificially synthesized primer sequence, p29 29 gttggaagaa agtaaattgg gaa 23 30 21 DNA Artificial Sequence Artificially synthesized primer sequence, p36 30 gatactctca ttaggaggtc c 21 31 26 DNA Artificial Sequence Artificially synthesized primer sequence, p48 31 cattaattga agtgagattt ttctgg 26 32 22 DNA Artificial Sequence Artificially synthesized primer sequence, p59 32 agcatttgtg gaaatactct gg 22 33 24 DNA Artificial Sequence Artificially synthesized primer sequence, p85 33 aactttattt ctcctccaca tcag 24 34 21 DNA Artificial Sequence Artificially synthesized primer sequence, p86 34 gtgaatggct cagagacaag g 21 35 11204 DNA Homo sapiens 35 aggttcagag agactgtggg aatatggggg aattagaggc tatctgaggc tcttcaacac 60 aataacccaa gaagctattt aaatgctctt taaggtattt acataaatat tactattctc 120 attgtgcttt tattttgtgt tatcatgatt ataattgaag tgtctactgt tactgcctcc 180 tgatctttgc tagctatgga gcatggactg ggcttttaga gcagcagccc caaaggaacc 240 taaacattaa agcagagctg ccctcaatgg tttaacctgt gtgactctgc ctatgacagc 300 cccacccacc catcttcact ggatccaaat caggagcaag gccgttgggg tacctggtgg 360 gggtgatgct gtcaggggag gagcccaaaa gggcaagctc aaatttgaat gtgaagggcc 420 aatgcactgt cagactgaga cagagaacca tcattaattg aagtgagatt tttctggcct 480 gagacttgca gggaggcaag aagacactct ggacaccact atggacaggt aaagaggcag 540 tcttctcgtg ggtgattgca ctggccttcc tctcagagca aatctgagta atgagactgg 600 tagctatccc tttctctcat gtaactgtct gactgataag atcagcttga tcaatatgca 660 tatatatttt ttgatctgtc tccttttctt ctattcagat cttatacgct gtcagcccaa 720 ttctttctgt ttcagacttc tcttgatttc cctctttttc atgtggcaaa agaagtagtg 780 cgtacaatgt actgattcgt cctgagattt gtaccatggt tgaaactaat ttatggtaat 840 aatattaaca tagcaaatct ttagagactc aaatcatgaa aaggtaatag cagtactgta 900 ctaaaaacgg tagtgctaat tttcgtaata attttgtaaa tattcaacag taaaacaact 960 tgaagacaca ctttcctagg gaggcgttac tgaaataatt tagctatagt aagaaaattt 1020 gtaattttag aaatgccaag cattctaaat taattgcttg aaagtcacta tgattgtgtc 1080 cattataagg agacaaattc attcaagcaa gttatttaat gttaaaggcc caattgttag 1140 gcagttaatg gcacttttac tattaactaa tctttccatt tgttcagacg tagcttaact 1200 tacctcttag gtgtgaattt ggttaaggtc ctcataatgt ctttatgtgc agtttttgat 1260 aggttattgt catagaactt attctattcc tacatttatg attactatgg atgtatgaga 1320 ataacaccta atccttatac tttacctcaa tttaactcct ttataaagaa cttacattac 1380 agaataaaga ttttttaaaa atatattttt ttgtagagac agggtcttag cccagccgag 1440 gctggtctct aagtcctggc ccaagcgatc ctcctgcctg ggcctcctaa agtgctggaa 1500 ttatagacat gagccatcac atccaatata cagaataaag atttttaatg gaggatttaa 1560 tgttcttcag aaaattttct tgaggtcaga caatgtcaaa tgtctcctca gtttacactg 1620 agattttgaa aacaagtctg agctataggt ccttgtgaag ggtccattgg aaatacttgt 1680 tcaaagtaaa atggaaagca aaggtaaaat cagcagttga aattcagaga aagacagaaa 1740 aggagaaaag atgaaattca acaggacaga agggaaatat attatcatta aggaggacag 1800 tatctgtaga gctcattagt gatggcaaaa tgacttggtc aggattattt ttaacccgct 1860 tgtttctggt ttgcacggct ggggatgcag ctagggttct gcctcaggga gcacagctgt 1920 ccagagcagc tgtcagcctg caagcctgaa acactccctc ggtaaagtcc ttcctactca 1980 ggacagaaat gacgagaaca gggagctgga aacaggcccc taaccagaga agggaagtaa 2040 tggatcaaca aagttaacta gcaggtcagg atcacgcaat tcatttcact ctgactggta 2100 acatgtgaca gaaacagtgt aggcttattg tattttcatg tagagtagga cccaaaaatc 2160 cacccaaagt cctttatcta tgccacatcc ttcttatcta tacttccagg acactttttc 2220 ttccttatga taaggctctc tctctctcca cacacacaca cacacacaca cacacacaca 2280 cacacacaca cacaaacaca caccccgcca accaaggtgc atgtaaaaag atgtagattc 2340 ctctgccttt ctcatctaca cagcccagga gggtaagtta atataagagg gatttattgg 2400 taagagatga tgcttaatct gtttaacact gggcctcaaa gagagaattt cttttcttct 2460 gtacttatta agcacctatt atgtgttgag cttatatata caaagggtta ttatatgcta 2520 atatagtaat agtaatggtg gttggtacta tggtaattac cataaaaatt attatccttt 2580 taaaataaag ctaattatta ttggatcttt tttagtattc attttatgtt ttttatgttt 2640 ttgatttttt aaaagacaat ctcaccctgt tacccaggct ggagtgcagt ggtgcaatca 2700 tagctttctg cagtcttgaa ctcctgggct caagcaatcc tcctgccttg gcctcccaaa 2760 gtgttgggat acagtcatga gccactgcat ctggcctagg atccatttag attaaaatat 2820 gcattttaaa ttttaaaata atatggctaa tttttacctt atgtaatgtg tatactggta 2880 ataaatctag tttgctgcct aaagtttaaa gtgctttcca ataagcttca tgtacgtgag 2940 gggagacatt taaagtgaaa cagacagcca ggtgtggtgg ctcacgcctg taatcccagc 3000 actctgggag gctgaggtgg gtggatcgct tgagccctgg agttcaagac cagcctgagc 3060 aacatggcaa aaccctgttt ctataacaaa aattagccgg gcatggtggc atgtgcctgt 3120 ggtcccagct actagggggc tgaggcagga gaatctttgg agcccaggag gtcaaggctg 3180 cactgagcag tgcttgcgcc actgcactcc agcctgggtg acaggaccag accttgcctc 3240 aaaaaaataa gaagaaaaat taaaaataaa tggaaacaac tacaaagagc tgttgtccta 3300 gatgagctac ttagttaggc tgatattttg gtatttaact tttaaagtca gggtctgtca 3360 cctgcactac attattaaaa tatcaattct caatgtatat ccacacaaag actggtacgt 3420 gaatgttcat agtaccttta ttcacaaaac cccaaagtag agactatcca aatatccatc 3480 aacaagtgaa caaataaaca aaatgtgcta tatccatgca atggaatacc accctgcagt 3540 acaaaggaag aagctacttg gggatgaatc ccaaagtcat gacgctaaat gaaagagtca 3600 gacatgaagg aggagataat gtatgccata cgaaattcta gaaaatgaaa gtaacttata 3660 gttacagaaa gcaaatcagg gcaggcatag aggctcacac ctgtaatccc agcactttga 3720 gaggccacgt gggaagattg ctagaactca ggagttcaag accagcctgg gcaacacagt 3780 gaaactccat tctccacaaa aatgggaaaa aaagaaagca aatcagtggt tgtcctgtgg 3840 ggaggggaag gactgcaaag agggaagaag ctctggtggg gtgagggtgg tgattcaggt 3900 tctgtatcct gactgtggta gcagtttggg gtgtttacat ccaaaaatat tcgtagaatt 3960 atgcatctta aatgggtgga gtttactgta tgtaaattat acctcaatgt aagaaaaaat 4020 aatgtgtaag aaaagtttca attctcttgc cagcaaacgt tattcaaatt cctgagccct 4080 ttacttcgca aattctctgc acttctgccc cgtaccatta ggtgacagca ctagctccac 4140 aaattggata aatgcatttc tggaaaagac tagggacaaa atccaggcat cacttgtgct 4200 ttcatatcaa ccacgctgta cagcttgtgt tgctgtctgc agctgcaatg gggactcttg 4260 atttctttaa ggaaacttgg gttaccagag tatttccaca aatgctattc aaattagtgc 4320 ttatgatatg caagacactg tgctaggagc cagaaaacaa agaggaggag aaatcagtca 4380 ttatgtggga acaacatagc aagatattta gatcattttg actagttaaa aaagcagcag 4440 agtacaaaat cacacatgca atcagtataa tccaaatcat gtaaatatgt gcctgtagaa 4500 agactagagg aataaacaca agaatcttaa cagtcattgt cattagacac taagtctaat 4560 tattattatt agacactatg atatttgaga tttaaaaaat ctttaatatt ttaaaattta 4620 gagctcttct atttttccat agtattcaag tttgacaatg atcaagtatt actctttctt 4680 tttttttttt tttttttttt tttgagatgg agttttggtc ttgttgccca tgctggagtg 4740 gaatggcatg accatagctc actgcaacct ccacctcctg ggttcaagca aagctgtcgc 4800 ctcagcctcc cgggtagatg ggattacagg cgcccaccac cacactcggc taatgtttgt 4860 atttttagta gagatggggt ttcaccatgt tggccaggct ggtctcaaac tcctgacctc 4920 agaggatcca cctgcctcag cctcccaaag tgctgggatt acagatgtag gccactgcgc 4980 ccggccaagt attgctctta tacattaaaa aacaggtgtg agccactgcg cccagccagg 5040 tattgctctt atacattaaa aaataggccg gtgcagtggc tcacgcctgt aatcccagca 5100 ctttgggaag ccaaggcggg cagaacaccc gaggtcagga gtccaaggcc agcctggcca 5160 agatggtgaa accccgtctc tattaaaaat acaaacatta cctgggcatg atggtgggcg 5220 cctgtaatcc cagctactca ggaggctgag gcaggaggat ccgcggagcc tggcagatct 5280 gcctgagcct gggaggttga ggctacagta agccaagatc atgccagtat acttcagcct 5340 gggcgacaaa gtgagaccgt aacaaaaaaa aaaaaattta aaaaaagaaa tttagatcaa 5400 gatccaactg taaaaagtgg cctaaacacc acattaaaga gtttggagtt tattctgcag 5460 gcagaagaga accatcaggg ggtcttcagc atgggaatgg catggtgcac ctggtttttg 5520 tgagatcatg gtggtgacag tgtggggaat gttattttgg agggactgga ggcagacaga 5580 ccggttaaaa ggccagcaca acagataagg aggaagaaga tgagggcttg gaccgaagca 5640 gagaagagca aacagggaag gtacaaattc aagaaatatt ggggggtttg aatcaacaca 5700 tttagatgat taattaaata tgaggactga ggaataagaa atgagtcaag gatggttcca 5760 ggctgctagg ctgcttacct gaggtggcaa agtcgggagg agtggcagtt taggacaggg 5820 ggcagttgag gaatattgtt ttgatcattt tgagtttgag gtacaagttg gacacttagg 5880 taaagactgg aggggaaatc tgaatataca attatgggac tgaggaacaa gtttatttta 5940 ttttttgttt cgttttcttg ttgaagaaca aatttaattg taatcccaag tcatcagcat 6000 ctagaagaca gtggcaggag gtgactgtct tgtgggtaag ggtttggggt ccttgatgag 6060 tatctctcaa ttggccttaa atataagcag gaaaaggagt ttatgatgga ttccaggctc 6120 agcagggctc aggagggctc aggcagccag cagaggaagt cagagcatct tctttggttt 6180 agcccaagta atgacttcct taaaaagctg aaggaaaatc cagagtgacc agattataaa 6240 ctgtactctt gcattttctc tccctcctct cacccacagc ctcttgatga accggaggaa 6300 gtttctttac caattcaaaa atgtccgctg ggctaagggt cggcgtgaga cctacctgtg 6360 ctacgtagtg aagaggcgtg acagtgctac atccttttca ctggactttg gttatcttcg 6420 caataaggta tcaattaaag tcagctttgc aagcagttta atggtcaact gtgagtgctt 6480 ttagagccac ctgctgatgg tattacttcc atcctttttt ggcatttgtg tctctatcac 6540 attcctcaaa tccttttttt tatttctttt tccatgtcca tgcacccata ttagacatgg 6600 cccaaaatat gtgatttaat tcctccccag taatgctggg caccctaata ccactccttc 6660 cttcagtgcc aagaacaact gctcccaaac tgtttaccag ctttcctcag catctgaatt 6720 gcctttgaga ttaattaagc taaaagcatt tttatatggg agaatattat cagcttgtcc 6780 aagcaaaaat tttaaatgtg aaaaacaaat tgtgtcttaa gcatttttga aaattaagga 6840 agaagaattt gggaaaaaat taacggtggt tcaattctgt tttccaaatg atttcttttc 6900 cctcctactc acatgggtcg taggccagtg aatacattca acatggtgat ccccagaaaa 6960 ctcagagaag cctcggctga tgattaatta aattgatctt tcggctaccc gagagaatta 7020 catttccaag agacttcttc accaaaatcc agatgggttt acataaactt ctgcccatgg 7080 gtatctcctc tctcctaaca cgctgtgacg tctgggcttg gtggaatctc agggaagcat 7140 ccgtggggtg gaaggtcatc gtctggctcg ttgtttgatg gttatattac catgcaattt 7200 tctttgccta catttgtatt gaatacatcc caatctcctt cctattcggt gacatgacac 7260 attctatttc agaaggcttt gattttatca agcactttca tttacttctc atggcagtgc 7320 ctattacttc tcttacaata cccatctgtc tgctttacca aaatctattt ccccttttca 7380 gatcctccca aatggtcctc ataaactgtc ctgcctccac ctagtggtcc aggtatattt 7440 ccacaatgtt acatcaacag gcacttctag ccattttcct tctcaaaagg tgcaaaaagc 7500 aacttcataa acacaaatta aatcttcggt gaggtagtgt gatgctgctt cctcccaact 7560 cagcgcactt cgtcttcctc attccacaaa aacccatagc cttccttcac tctgcaggac 7620 tagtgctgcc aagggttcag ctctacctac tggtgtgctc ttttgagcaa gttgcttagc 7680 ctctctgtaa cacaaggaca atagctgcaa gcatccccaa agatcattgc aggagacaat 7740 gactaaggct accagagccg caataaaagt cagtgaattt tagcgtggtc ctctctgtct 7800 ctccagaacg gctgccacgt ggaattgctc ttcctccgct acatctcgga ctgggaccta 7860 gaccctggcc gctgctaccg cgtcacctgg ttcacctcct ggagcccctg ctacgactgt 7920 gcccgacatg tggccgactt tctgcgaggg aaccccaacc tcagtctgag gatcttcacc 7980 gcgcgcctct acttctgtga ggaccgcaag gctgagcccg aggggctgcg gcggctgcac 8040 cgcgccgggg tgcaaatagc catcatgacc ttcaaaggtg cgaaagggcc ttccgcgcag 8100 gcgcagtgca gcagcccgca ttcgggattg cgatgcggaa tgaatgagtt agtggggaag 8160 ctcgagggga agaagtgggc ggggattctg gttcacctct ggagccgaaa ttaaagatta 8220 gaagcagaga aaagagtgaa tggctcagag acaaggcccc gaggaaatga gaaaatgggg 8280 ccagggttgc ttctttcccc tcgatttgga acctgaactg tcttctaccc ccatatcccc 8340 gccttttttt cctttttttt ttttttgaag attattttta ctgctggaat acttttgtag 8400 aaaaccacga aagaactttc aaagcctggg aagggctgca tgaaaattca gttcgtctct 8460 ccagacagct tcggcgcatc cttttggtaa ggggcttcct cgctttttaa attttctttc 8520 tttctctaca gtcttttttg gagtttcgta tatttcttat attttcttat tgttcaatca 8580 ctctcagttt tcatctgatg aaaactttat ttctcctcca catcagcttt ttcttctgct 8640 gtttcaccat tcagagccct ctgctaaggt tccttttccc tcccttttct ttcttttgtt 8700 gtttcacatc tttaaatttc tgtctctccc cagggttgcg tttccttcct ggtcagaatt 8760 cttttctcct tttttttttt tttttttttt ttttttaaac aaacaaacaa aaaacccaaa 8820 aaaactcttt cccaatttac tttcttccaa catgttacaa agccatccac tcagtttaga 8880 agactctccg gccccaccga cccccaacct cgttttgaag ccattcactc aatttgcttc 8940 tctctttctc tacagcccct gtatgaggtt gatgacttac gagacgcatt tcgtactttg 9000 ggactttgat agcaacttcc aggaatgtca cacacgatga aatatctctg ctgaagacag 9060 tggataaaaa acagtccttc aagtcttctc tgtttttatt cttcaactct cactttctta 9120 gagtttacag aaaaaatatt tatatacgac tctttaaaaa gatctatgtc ttgaaaatag 9180 agaaggaaca caggtctggc cagggacgtg ctgcaattgg tgcagttttg aatgcaacat 9240 tgtcccctac tgggaataac agaactgcag gacctgggag catcctaaag tgtcaacgtt 9300 tttctatgac ttttaggtag gatgagagca gaaggtagat cctaaaaagc atggtgagag 9360 gatcaaatgt ttttatatca acatccttta ttatttgatt catttgagtt aacagtggtg 9420 ttagtgatag atttttctat tcttttccct tgacgtttac tttcaagtaa cacaaactct 9480 tccatcaggc catgatctat aggacctcct aatgagagta tctgggtgat tgtgacccca 9540 aaccatctct ccaaagcatt aatatccaat catgcgctgt atgttttaat cagcagaagc 9600 atgtttttat gtttgtacaa aagaagattg ttatgggtgg ggatggaggt atagaccatg 9660 catggtcacc ttcaagctac tttaataaag gatcttaaaa tgggcaggag gactgtgaac 9720 aagacaccct aataatgggt tgatgtctga agtagcaaat cttctggaaa cgcaaactct 9780 tttaaggaag tccctaattt agaaacaccc acaaacttca catatcataa ttagcaaaca 9840 attggaagga agttgcttga atgttgggga gaggaaaatc tattggctct cgtgggtctc 9900 ttcatctcag aaatgccaat caggtcaagg tttgctacat tttgtatgtg tgtgatgctt 9960 ctcccaaagg tatattaact atataagaga gttgtgacaa aacagaatga taaagctgcg 10020 aaccgtggca cacgctcata gttctagctg cttgggaggt tgaggaggga ggatggcttg 10080 aacacaggtg ttcaaggcca gcctgggcaa cataacaaga tcctgtctct caaaaaaaaa 10140 aaaaaaaaaa agaaagagag agggccgggc gtggtggctc acgcctgtaa tcccagcact 10200 ttgggaggcc gagccgggcg gatcacctgt ggtcaggagt ttgagaccag cctggccaac 10260 atggcaaaac cccgtctgta ctcaaaatgc aaaaattagc caggcgtggt agcaggcacc 10320 tgtaatccca gctacttggg aggctgaggc aggagaatcg cttgaaccca ggaggtggag 10380 gttgcagtaa gctgagatcg tgccgttgca ctccagcctg ggcgacaaga gcaagactct 10440 gtctcagaaa aaaaaaaaaa aaagagagag agagagaaag agaacaatat ttgggagaga 10500 aggatgggga agcattgcaa ggaaattgtg ctttatccaa caaaatgtaa ggagccaata 10560 agggatccct atttgtctct tttggtgtct atttgtccct aacaactgtc tttgacagtg 10620 agaaaaatat tcagaataac catatccctg tgccgttatt acctagcaac ccttgcaatg 10680 aagatgagca gatccacagg aaaacttgaa tgcacaactg tcttatttta atcttattgt 10740 acataagttt gtaaaagagt taaaaattgt tacttcatgt attcatttat attttatatt 10800 attttgcgtc taatgatttt ttattaacat gatttccttt tctgatatat tgaaatggag 10860 tctcaaagct tcataaattt ataactttag aaatgattct aataacaacg tatgtaattg 10920 taacattgca gtaatggtgc tacgaagcca tttctcttga tttttagtaa acttttatga 10980 cagcaaattt gcttctggct cactttcaat cagttaaata aatgataaat aattttggaa 11040 gctgtgaaga taaaatacca aataaaataa tataaaagtg atttatatga agttaaaata 11100 aaaaatcagt atgatggaat aaacttgaga gtccagaagt tatcccatac atctgtaatc 11160 aactaatttc tcacaagggt gtaaggacca ttcaatggag aaaa 11204 36 229 PRT Mus musculus 36 Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg 1 5 10 15 Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu 20 25 30 Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His 35 40 45 Ser Val Trp Arg His Thr Ser Gln Asn Thr Ser Asn His Val Glu Val 50 55 60 Asn Phe Leu Glu Lys Phe Thr Thr Glu Arg Tyr Phe Arg Pro Asn Thr 65 70 75 80 Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys 85 90 95 Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg His Pro Tyr Val Thr Leu 100 105 110 Phe Ile Tyr Ile Ala Arg Leu Tyr His His Thr Asp Gln Arg Asn Arg 115 120 125 Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met 130 135 140 Thr Glu Gln Glu Tyr Cys Tyr Cys Trp Arg Asn Phe Val Asn Tyr Pro 145 150 155 160 Pro Ser Asn Glu Ala Tyr Trp Pro Arg Tyr Pro His Leu Trp Val Lys 165 170 175 Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys 180 185 190 Leu Lys Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile 195 200 205 Thr Leu Gln Thr Cys His Tyr Gln Arg Ile Pro Pro His Leu Leu Trp 210 215 220 Ala Thr Gly Leu Lys 225 

What is claimed is:
 1. An isolated nucleic acid encoding a protein comprising the amino acid sequence of SEQ ID NO:2 or 8 or a functional fragment thereof.
 2. The isolated nucleic acid of claim 1, wherein the protein has a cytidine deaminase activity.
 3. An isolated nucleic acid or a fragment thereof comprising the nucleotide sequence of SEQ ID NO:1 or
 7. 4. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) nucleotides 93 to 689 of SEQ ID NO:1; and (b) nucleotides 80 to 676 of SEQ ID NO:7.
 5. An isolated nucleic acid selected from the group consisting of: (a) a nucleic acid that (i) hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:1, and (ii) encodes a polypeptide having a cytidine deaminase activity; (b) a nucleic acid that (i) hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO:7, and (ii) encodes a polypeptide having a cytidine deaminase activity; and (c) a fragment of (a) or (b).
 6. A substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 8 or a fragment thereof that (a) has a cytidine deaminase activity activity or (b) is immunogenic.
 7. A substantially pure polypeptide comprising substantially the same amino acid sequence as that of SEQ ID NO:2 or 8 and having a cytidine deaminase activity.
 8. A substantially pure polypeptide selected from the group consisting of: (a) a mammalian polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to the sequence of SEQ ID NO:1, wherein the polypeptide has a cytidine deaminase activity; and (b) a mammalian polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to the sequence of SEQ ID NO:7, wherein the polypeptide has a cytidine deaminase activity.
 9. An expression vector comprising the nucleic acid of claim
 1. 10. An expression vector comprising the nucleic acid of claim
 2. 11. An expression vector comprising the nucleic acid of claim
 3. 12. An expression vector comprising the nucleic acid of claim
 4. 13. An expression vector comprising the nucleic acid of claim
 5. 14. A cultured host cell comprising the expression vector of claim
 9. 15. A cultured host cell comprising the expression vector of claim
 10. 16. A cultured host cell comprising the expression vector of claim
 11. 17. A cultured host cell comprising the expression vector of claim
 12. 18. A cultured host cell comprising the expression vector of claim
 13. 19. An antibody or a reactive portion thereof to the polypeptide of claim
 6. 20. An antibody or a reactive portion thereof to the polypeptide of claim
 7. 21. An antibody or a portion thereof reactive to the polypeptide, of claim
 8. 22. The antibody of claim 21, wherein the antibody is a monoclonal antibody.
 23. A pharmaceutical composition comprising the antibody or the portion thereof of claim 21 and a pharmaceutically acceptable carrier.
 24. A pharmaceutical composition comprising the antibody or the portion thereof of claim 22 and a pharmaceutically acceptable carrier.
 25. A cell producing a monoclonal antibody reactive to the polypeptide of claim
 6. 26. A cell producing a monoclonal antibody reactive to the polypeptide of claim
 7. 27. A cell producing a monoclonal antibody reactive to the polypeptide of claim
 8. 28. The cell of claim 25, wherein the cell is a hybridoma obtained by fusing, with a mammalian myeloma cell, a non-human mammalian B cell that produces a monoclonal antibody.
 29. The cell of claim 26, wherein the cell is a hybridoma obtained by fusing, with a mammalian myeloma cell, a non-human mammalian B cell that produces a monoclonal antibody.
 30. The cell of claim 27, wherein the cell is a hybridoma obtained by fusing, with a mammalian myeloma cell, a non-human mammalian B cell that produces a monoclonal antibody.
 31. The cell of claim 25, wherein the cell is a transgenic cell transformed by introducing, into the cell, either or both of a nucleic acid encoding a heavy chain of the monoclonal antibody and a nucleic acid encoding a light chain of the monoclonal antibody.
 32. The cell of claim 26, wherein the cell is a transgenic cell transformed by introducing, into the cell, either or both of a nucleic acid encoding a heavy chain of the monoclonal antibody and a nucleic acid encoding a light chain of the monoclonal antibody.
 33. The cell of claim 27, wherein the cell is a transgenic cell transformed by introducing, into the cell, either or both of a nucleic acid encoding a heavy chain of the monoclonal antibody and a nucleic acid encoding a light chain of the monoclonal antibody.
 34. An isolated nucleic acid comprising the nucleotide sequence of (a) SEQ ID NO:9, (b) SEQ ID NO:10, or (c) SEQ ID NO:35.
 35. An isolated nucleic acid comprising the nucleotide sequence of (a) SEQ ID NO:11, (b) SEQ ID NO:12, (c) SEQ ID NO:13, (d) SEQ ID NO:14, or (e) SEQ ID NO:15.
 36. An isolated nucleic acid comprising a sequence complementary to any one of: (a) SEQ ID NO:9, (b) SEQ ID NO:10, (c) SEQ ID NO:11, (d) SEQ ID NO:12, (e) SEQ ID NO:13, (f) SEQ ID NO:14, (g) SEQ ID NO:15, or (h) SEQ ID NO:35.
 37. An isolated oligonucleotide comprising a sequence complementary to a fragment of any one of: (a) SEQ ID NO:9, (b) SEQ ID NO:10, (c) SEQ ID NO:11, (d) SEQ ID NO:12, (e) SEQ ID NO:13, (f) SEQ ID NO:14, (g) SEQ ID NO:15, or (h) SEQ ID NO:35.
 38. The nucleic acid of claim 37, wherein the primer comprises a nucleotide sequence of any one of: (a) SEQ ID NO:18, (b) SEQ ID NO:19, (c) SEQ ID NO:20, (d) SEQ ID NO:21, (e) SEQ ID NO:22, (f) SEQ ID NO:23, (g) SEQ ID NO:24, (h) SEQ ID NO:25, (i) SEQ ID NO:26, (j) SEQ ID NO:27, (k) SEQ ID NO:28, (l) SEQ ID NO:29, (m) SEQ ID NO:30, (n) SEQ ID NO:31, (o) SEQ ID NO:32, (p) SEQ ID NO:33, or (q) SEQ ID NO:34.
 39. A pair of isolated oligonucleotide primers selected from the group consisting of: (a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:31 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:32, (b) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:20 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:22, (c) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:21 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:30, (d) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:24 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:25, (e) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:23 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:27, (f) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:23 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:28, (g) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:23 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29 (h) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:26 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:27, (i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:26 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:28, (j) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:26 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29, (k) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:34 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:28, (l) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:34 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29, (m) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:33 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:29, or (n) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:18 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:19.
 40. A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of: (a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein, and (b) (i) comparing the level of the AID protein produced by the cells cultured in the presence of the substance with the level of the AID protein produced by the cells cultured in the absence of the substance, or (ii) comparing the level of the AID protein-encoding mRNA transcribed in the cells cultured in the presence of the substance with the level of the AID protein-encoding mRNA transcribed in the cells cultured in the absence of the substance.
 41. A method for identifying a substance that regulates transcription of a gene encoding an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8 into mRNA, or production of the AID protein, the method comprising the steps of: (a) culturing, separately in the presence and the absence of the substance, cells producing the AID protein and a second protein other than the AID protein, wherein transcription of a gene encoding the second protein into mRNA is dependent in the cells on the degree of transcription of the gene encoding the AID protein into mRNA, and (b) comparing the level of the second protein produced by the cells cultured in the presence of the substance with the level of the second protein produced by the cells cultured in the absence of the substance.
 42. The method of claim 40, wherein the cells are transgenic cells transformed with a gene encoding the AID protein.
 43. The method of claim 41, wherein the cells are transgenic cells transformed with a gene encoding the AID protein.
 44. The method of claim 41, wherein the cells are transgenic cells transformed with a gene encoding the AID protein and a gene encoding the second protein.
 45. The method of claim 41, wherein the second protein is a reporter protein.
 46. The method of claim 45, wherein comparison of the level of the second protein is comparison of the level of a signal generated by the reporter protein.
 47. The method of claim 45, wherein the reporter protein is luciferase.
 48. A method for identifying a substance that inhibits an enzyme activity of an AID protein comprising the amino acid sequence of SEQ ID NO:2 or 8, the method comprising the step of: (a) culturing, separately in the presence and the absence of the substance, mammal-derived B cells or tissues comprising the B cells, and comparing enzyme activities of the AID proteins in the B cells separately cultured, or (b) (i) administering the substance separately to an AID) gene knockout mouse whose endogenous AID gene is inactivated so that transcription of the endogenous AID gene into mRNA is inhibited, and to a normal mouse, and (ii) comparing enzyme activities of the AID proteins in the B cells isolated from the respective mice.
 49. The method of claim 48, wherein the enzyme activity is a cytidine deaminase activity. 